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

Abstract

The invention discloses a cell surface marker for detecting circulating tumor cells in breast cancer patients and its application. The cell surface markers include TSPAN1, FAIM2, KCNJ4, GABRD, PDK1L2, OR1OK1, UPK1B and OR51M1; using cell surface markers The corresponding antibodies perform immune capture of breast cancer CTCs; the present invention utilizes the obtained surface markers of circulating tumor cells of breast cancer patients to obtain the best antibody combination for immune enrichment screening and improves the enrichment and capture efficiency of breast cancer CTCs. , effectively improves the specificity and sensitivity of CTC capture, and has the effect of efficiently capturing breast cancer CTCs. It overcomes the problems of insufficient types, low detection rate and low purity of existing breast cancer CTC enrichment markers.

Description

Cell surface markers and applications for detection of circulating tumor cells in breast cancer patients

Technical field

The present invention relates to the technical field of tumor cell detection, specifically to cell surface markers and applications for detecting CTC in breast cancer patients.

Background technique

Circulating tumor cells (CTC) refer to tumor cells that are detached from tumor tissue and released into the blood circulation system. They can truly represent tumor load and characteristics, dynamically reflect tumor genomic information and gene expression profile changes, and are related to the characteristics of patients' tumors. Distant metastasis is closely related and is of great significance for the diagnosis and clinical staging of malignant tumors. At present, CTC research has achieved remarkable results in early tumor screening, dynamic monitoring, and immunotherapy selection. CTC detection has become one of the most promising non-invasive tumor diagnosis and treatment methods. The field of liquid biopsy to which it belongs has been rated as one of the top ten future medical technologies by MIT technology review. Compared with commonly used tumor diagnosis methods such as imaging evidence and tissue biopsy, CTC detection has the advantages of early stage, real-time efficiency, non-invasiveness, convenient sample collection and repeatable monitoring. Compared with conventional tumor marker detection, CTC detection can usually reflect changes in the disease earlier and can more sensitively monitor dynamic changes in tumors to evaluate therapeutic efficacy, recurrence and metastasis monitoring, and targeted drug companion diagnostics during the treatment process. It has important guiding significance in the formulation of clinical treatment plans. At present, CTC has been officially included in the tumor staging system, and has become another breast cancer prognosis evaluation tool after the four biological indicators of ER/PR, Her-2, Ki-67 and tumor histological grade. In 2019, CTC was included in the Chinese Breast Cancer Diagnosis and Treatment Guidelines for the first time, and it was clearly stated that CTC can reflect the condition of tumor tissue, and can be used non-invasively to replace tissue samples for pathological diagnosis, disease monitoring, molecular sequencing, etc., not only for dynamic monitoring, but also for After the judgment is healed.

However, due to the special properties of CTCs, there are still many problems and challenges in the clinical application of CTC detection. At present, CTC detection methods are mainly based on the physical properties and biological characteristics of CTC. Including cell filtration method, density gradient centrifugation method, dielectrophoresis separation method, etc., CTC detection is mainly based on biophysical characteristics such as cell size, density, deformability, and charge characteristics. However, due to the rarity and heterogeneity of CTCs, the non-uniform cell size, and the certain flexibility of the cells themselves, the above method lacks specificity when isolating CTCs, resulting in problems such as low purity and low efficiency.

Currently, the most widely used is CTC detection technology based on immunoaffinity, which uses antibodies, nucleic acid aptamers or peptides bound to an appropriate matrix (such as magnetic beads) to bind to the CTC surface target through magnetic force or other forces. Positive or negative selection achieves CTC enrichment. The negative selection method uses specific antigens on the surface of leukocytes (mainly CD45) to remove leukocytes from the blood and then enrich CTCs. It has the characteristics of mature antibodies, easy acquisition, and high efficiency of capturing leukocytes. However, this method has certain limitations and requires lysis of red blood cells. , Antibodies are prone to non-specific binding to CTCs in poor condition, resulting in low purity of CTC detection and easy to lead to false positive results. The positive selection method, that is, the immunoenrichment method, directly enriches the corresponding antibodies to capture CTCs through certain special biomarkers on the surface of CTCs, such as epithelial cell adhesion molecules (EpCAM). This method is not sensitive to the state of CTC, has high detection purity, is simple to operate, can be automated, and is durable. Currently, the CellSearch detection system approved by the U.S. Food and Drug Administration (FDA) and the CellCollector detection system approved by my country's CFDA are both immunoenrichment methods. It should be noted that CTCs are highly heterogeneous, and there are certain differences in markers on the surface of CTCs in different types of tumors. Traditional detection systems used to capture CTCs do not fully take into account the heterogeneity of CTCs and tumor specificity, and use a relatively single marker or a combination of markers to enrich and screen CTCs. For example, currently, the markers on the surface of CTCs used in immunoenrichment methods are mainly the adhesion molecule EpCAM and cytokeratin. This fixed antibody combination is obviously not applicable to all types of tumors, especially CTCs lacking the above-mentioned surface markers, resulting in missed detection.

Contents of the invention

In view of the problems existing in the prior art, the present invention provides a cell surface marker for detecting CTC in breast cancer patients and its application.

The technical solution adopted by the present invention is:

Cell surface markers used to detect circulating tumor cells in breast cancer patients, including TSPAN1, FAIM2, KCNJ4, GABRD, PDK1L2, OR1OK1, UPK1B, and OR51M1.

For the application of cell surface markers for detecting CTCs in breast cancer patients, antibodies corresponding to cell surface markers are used to immunocapture breast cancer CTCs.

Further, HER2 antibody, EpCam antibody, FAIM2 antibody and GABRD antibody are used together, or HER2 antibody, EpCam antibody, KCNJ4 antibody and GABRD antibody are used together.

Further, the application of antibodies corresponding to cell surface markers in the preparation of reagents and/or drugs for detecting and/or treating breast cancer.

Further, the process of immune capture of breast cancer CTC cells is as follows:

Step 1: Coat the microfluidic chip with cell surface marker antibodies;

Step 2: Flow the cultured breast cancer cell suspension or breast cancer patient blood sample through the chip at a certain speed;

Step 3: Use the collection solution to collect the cells captured by the chip and count them; the count represents the ability of the antibody to capture breast cancer cells.

The screening method for cell surface markers for detecting CTCs in breast cancer patients includes the following steps:

Based on the RNA-seq and scRNA-seq sequencing data of circulating tumor cell CTC samples of breast cancer patients in the public database, the specific expression profile information and heterogeneity characteristics of CTCs in breast cancer patients were analyzed, and CTC highly expressed genes were obtained;

Filter according to the gene expression abundance information of other cell types in the blood sample and combine it with the surface protein expression characteristic information to obtain CTC-specific surface markers;

The obtained CTC-specific surface markers were compared with the expression profiles of breast cancer patient tumor tissue samples in the public database to determine the cell surface markers of CTCs.

The beneficial effects of the present invention are:

(1) The marker in the present invention is a novel breast cancer CTC surface marker with enrichment ability;

(2) The present invention uses the surface markers of breast cancer CTCs obtained through screening to obtain the best antibody combination for immune enrichment screening, improves the enrichment and capture efficiency of breast cancer CTCs, and effectively improves the specificity and sensitivity of CTC capture. It is highly efficient in capturing breast cancer CTCs;

(3) The present invention overcomes the existing problems of insufficient types of breast cancer CTC enrichment markers, low detection rate, and low purity, achieves efficient capture of breast cancer CTCs, and promotes CTC detection in the auxiliary diagnosis of breast cancer and monitoring of tumor dynamics. , application in clinical treatment and prognosis evaluation; providing more accurate information and strong support to achieve real-time precise individual treatment, and has broad application prospects and market value.

Description of the drawings

Figure 1 is a schematic diagram of breast cancer CTC surface marker screening. a is the expression abundance distribution of breast cancer CTC-specific expression markers in CTC RNA-seq samples; b is the total CTC-specific expression in breast cancer CTC RNA-seq and scRNA-seq data sets. The expression level of markers in CTC RNA-seq samples; c is the expression level of CTC-specific expression markers in CTC scRNA-seq samples shared by breast cancer CTC RNA-seq and scRNA-seq data sets; d is the specific expression of breast cancer CTCs Expression marker protein level expression specificity.

Figure 2 is a schematic diagram of protein sequence analysis of markers screened in the present invention.

Figure 3 is a schematic diagram of using a microfluidic chip to capture CTCs from blood samples in the present invention.

Figure 4 is a diagram showing the efficiency of enriching and screening breast cancer cells SKBR3 and MCF-7 with the marker antibodies screened in the present invention.

Figure 5 is a diagram showing the efficiency of enriching and screening breast cancer cells SKBR3 and MCF-7 using the combination of marker antibodies and EpCAM screened in the present invention.

Figure 6 is a diagram showing the efficiency of the combined enrichment screening of breast cancer cells SKBR3 and MCF-7 using the marker antibody and HER2 screened in the present invention.

Figure 7 is a diagram showing the efficiency of enriching and screening breast cancer cells SKBR3 and MCF-7 using the combination of marker antibodies and EpCAM+HER2 screened in the present invention.

Figure 8 is a graph showing the efficiency of enriching and screening breast cancer cells SKBR3 and MCF-7 using the combination of four antibodies (FAIM2, KCNJ4, GABRD, UPK1B) selected in the present invention and the combination of EpCAM+HER2.

Figure 9 is a graph showing the enrichment and screening efficiency of the selected combined antibodies of the present invention on four types of breast cancer cells. (HER2+EpCAM, EpCAM+HER2+FAIM2+GABRD, HER2+EpCAM+KCNJ4+GABRD)

Figure 10 is a graph showing the enrichment and screening efficiency of the selected combined antibodies of the present invention on four types of breast cancer cells. (HER2+EpCAM, EpCAM+HER2+FAIM2+GABRD)

Figure 11 is a schematic diagram of the capture ability of the combined antibodies (EpCAM+HER2+FAIM2+GABRD) selected in the present invention on CTCs in the blood of breast cancer patients.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

A cell surface marker for detecting circulating tumor cells in breast cancer patients, including one or two or more of TSPAN1, FAIM2, KCNJ4, GABRD, PDK1L2, OR1OK1, UPK1B and OR51M1 mixed in any proportion.

The screening method includes the following steps:

Based on the RNA-seq and scRNA-seq sequencing data of circulating tumor cell CTC samples of breast cancer patients in the public database, the specific expression profile information and heterogeneity characteristics of CTCs in breast cancer patients were analyzed, and CTC highly expressed genes were obtained;

Filter according to the gene expression abundance information of other cell types in the blood sample and combine it with the surface protein expression characteristic information to obtain CTC-specific surface markers;

The obtained CTC-specific surface markers were compared with the expression profiles of breast cancer patient tumor tissue samples in the public database to determine the cell surface markers of CTCs, as shown in Figure 1.

After the above screening, candidate CTC-specific surface markers were initially selected, and further protein sequence analysis was performed on the genes obtained above, as shown in Figure 2. From these markers, markers with transmembrane segments and extracellular segments and relatively high expression levels were screened for testing. The selected genes in Figure 2 include: TSPAN1, FAIM2, KCNJ4, GABRD, PDK1L2, OR1OK1, UPK1B and OR51M1.

Antibodies corresponding to the surface markers screened as shown in Figure 2 were used to conduct relevant tumor cell enrichment screening experiments, including breast cancer cell lines and blood samples from breast cancer patients. The ability of these surface markers to enrich and screen breast cancer tumor cells and CTCs in blood was experimentally verified.

The process of immune capture of breast cancer CTC cells is as follows: As shown in Figure 3:

Step 1: Coat the microfluidic chip with cell surface marker antibodies;

Step 2: Flow the cultured breast cancer cell suspension or breast cancer patient blood sample through the chip at a certain speed; the cell suspension is cells added to 7.5mL of healthy human whole blood, mixed and then separated with lymphocytes The collected cell pellets were then treated with 7.5 mL of protective solution to form a cell suspension.

Step 3: Use the collection solution to collect the cells captured by the chip and count them; the count represents the ability of the antibody to capture breast cancer cells.

Example 1

Prepare or purchase TSPAN1, FAIM2, KCNJ4, GABRD, PDK1L2, OR1OK1, UPK1B and OR51M1 antibodies, use these antibodies to coat the chip respectively, and test the ability of each antibody to enrich and screen breast cancer cells (the method is shown in Figure 3, not included blood sample). In this example, classic breast cancer cells SKBR3 and MCF-7 were selected as experimental cells. After the chip is coated, count the cultured SKBR3 and MCF-7 cells. Each group has 1,000 cells. They flow through the chip at a certain speed. Use the collection solution to collect the cells captured by the chip and count them. The capture ability of different antibodies on SKBR3 and MCF-7 breast cancer cells was counted. The results are shown in Figure 4. The y-axis represents the capture efficiency, which is the percentage of cells enriched and captured by a single antibody. The x-axis represents newly developed breast cancer CTC-specific expression surface marker antibodies.

As can be seen from the figure, the screened markers all have a certain ability to enrich and capture SKBR3 cells. The enrichment and capture efficiency from high to low is TSPAN1, FAIM2, KCNJ4, GABRD, PDK1L2, OR10K1, UPK1B and OR51M1. .

Example 2

The screened surface marker antibodies were combined with EpCam to test the ability of the superimposed antibodies to enrich and capture breast cancer tumor cells (the method is shown in Figure 3, excluding blood samples). The results are shown in Figure 5. The y-axis represents the capture efficiency, that is, 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 and corresponding antibodies is 1:1.

Breast cancer classic cells SKBR3 and MCF-7 were selected as experimental cells. After the chip is coated, count the cultured SKBR3 and MCF-7 cells. Each group has 1,000 cells. They flow through the chip at a certain speed. Use the collection solution to collect the cells captured by the chip and count them. The capture ability of different antibodies on SKBR3 and MCF-7 breast cancer cells was counted. It can be seen from the figure that the combined antibodies have good capture capabilities for breast cancer cells SKBR3 and MCF-7, and the capture capabilities are greater than the single EpCam antibody.

Example 3

The screened surface marker antibodies were combined with HER2 to test the ability of the superimposed antibody to enrich and capture breast cancer tumor cells (the method is shown in Figure 3, excluding blood samples). The results are shown in Figure 6. The y-axis represents the capture efficiency, that is, 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 corresponding antibodies is 1:1.

Breast cancer classic cells SKBR3 and MCF-7 were selected as experimental cells. After the chip is coated, count the cultured SKBR3 and MCF-7 cells. Each group has 1,000 cells. They flow through the chip at a certain speed. Use the collection solution to collect the cells captured by the chip and count them. The capture ability of different antibodies on SKBR3 and MCF-7 breast cancer cells was counted. It can be seen from the figure that the combined antibodies have good capture ability for SKBR3. The capture ability of the combined antibodies to MCF-7 was greater than that of a single HER2 antibody.

Example 4

The screened surface marker antibodies were combined with EpCam and HER2 to test the ability of the superimposed antibody enrichment to capture breast cancer tumor cells (the method is shown in Figure 3, excluding blood samples). The results are shown in Figure 7. The y-axis represents the capture efficiency, that is, the percentage of antibody-enriched captured cells. The x-axis represents EpCam and HER2 and in combination with other surface marker antibodies. The mass ratio of the corresponding antibodies is 1:1:1.

Breast cancer classic cells SKBR3 and MCF-7 were selected as experimental cells. After the chip is coated, count the cultured SKBR3 and MCF-7 cells. Each group has 1,000 cells. They flow through the chip at a certain speed. Use the collection solution to collect the cells captured by the chip and count them. The capture ability of different antibodies on SKBR3 and MCF-7 breast cancer cells was counted.

Through cell line experiments, the one with higher efficiency was selected. As can be seen from the figure, the combined antibodies have good capture ability for SKBR3. The capture ability of the combined antibodies to MCF-7 was greater than that of the EpCam and HER2 combination.

Example 5

The screened surface marker antibodies (FAIM2, KCNJ4, GABRD, UPK1B) were combined with EpCam and HER2 in pairs to test the ability of the superimposed antibody enrichment to capture breast cancer tumor cells (the method is shown in Figure 3, excluding blood samples) . The results are shown in Figure 8. The y-axis represents the capture efficiency, that is, the percentage of antibody-enriched captured cells. The x-axis represents EpCam and HER2 combined with other surface marker antibodies (FAIM2, KCNJ4, GABRD, UPK1B) in pairs. The mass ratio of the corresponding antibodies is 1:1:1:1.

Breast cancer classic cells SKBR3 and MCF-7 were selected as experimental cells. After the chip is coated, count the cultured SKBR3 and MCF-7 cells. Each group has 1,000 cells. They flow through the chip at a certain speed. Use the collection solution to collect the cells captured by the chip and count them. The 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: FAIM2, KCNJ4, GABRD, and UPK1B. These four antibodies were combined in pairs and combined with EpCam and HER2. As can be seen from the figure, its capture efficiency is significantly higher than the existing capture solutions of EpCam and HER2 alone or combined. After further optimization, it was found that the two most efficient combination schemes are HER2, EpCam, FAIM2 and GABRD and HER2, EpCam, KCNJ4 and GABRD. The capture efficiency can reach 95% and 93% respectively.

Example 6

The chip was coated with HER2, EpCam, FAIM2 and GABRD and HER2, EpCam, KCNJ4 and GABRD combination antibodies, and four breast cancer cells, SKBR3, BT474, MCF-7 and MDA-MB-231, were cultured. Each cell count was 1000, respectively. The 1,000 four types of breast cancer cells were flowed through the chip at a certain flow rate, and the cells captured by the chip were collected with a collection solution. The capturing ability of the combined antibodies on the four types of breast cancer cells was counted and calculated. As shown in Figure 9, the y-axis represents the capture efficiency, that is, the percentage of antibody-enriched captured cells. The x-axis represents breast cancer cell lines. The antibody combinations are: HER2+EpCam antibody combination, and the antibody mass ratio is 1:1. For HER2+EpCam+FAIM2+GABRD and HER2+EpCam+KCNJ4+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 is significantly higher than that of the HER2+EpCam antibody combination. Moreover, the HER2+EpCam+FAIM2+GABRD combination antibody has better ability to enrich and capture breast cancer cells than the HER2+EpCam+KCNJ4+GABRD combination antibody.

Example 7

Choose the HER2+EpCam+FAIM2+GABRD combination antibody-coated chip. Count 1,000 cells of each of the four cultured breast cancer cells, SKBR3, BT474, MCF-7 and MDA-MB-231. Add these 1,000 four breast cancer cells to 7.5 ml of healthy human whole blood and mix well. Afterwards, it is treated with lymphocyte separation solution. The collected cell pellet is then added with 7.5 ml of protective solution to form a cell suspension. The cell suspension flows through the chip at a certain flow rate. The cells captured by the chip are collected with the collection solution and fluorescent Marker is used to collect the cells. The cells were fluorescently stained. After scanning and counting, the chip's ability to capture four types of breast cancer cells in whole blood was calculated. The results are shown in Figure 10. The y-axis represents the capture efficiency, that is, the percentage of antibody-enriched captured cells. The x-axis represents breast cancer cell lines. The antibody combination is: HER2+EpCam antibody combination, and the antibody mass ratio is 1:1. HER2+EpCam+FAIM2+GABRD, the mass ratio of 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 is significantly higher than that of the HER2+EpCam antibody combination.

Example 8

Choose the HER2+EpCam+FAIM2+GABRD combination antibody-coated chip. Extract 7.5ml of whole blood from breast cancer patients and process it with red blood cell lysis solution. The collected cell pellet is then added with 7.5ml of protective solution to form a cell suspension. The cell suspension flows through the chip at a certain flow rate and is captured by the chip using the collection solution. cells, and fluorescently stain the collected cells with a fluorescent marker. Use a fluorescence scanning counter to scan and count the samples, and record the number of CTCs captured by the combined antibody-coated chip in the blood of breast cancer patients. The results are shown in Figure 11. The y-axis represents the capture efficiency, that is, the percentage of antibody-enriched captured cells. The x-axis represents breast cancer cell lines. The antibody combination is: HER2+EpCam antibody combination, and the antibody mass ratio is 1:1. HER2+EpCam+FAIM2+GABRD, the mass ratio of antibodies is 1:1:1:1.

As can be seen from the figure, the capture ability of the combined antibodies on four breast cancer cells is significantly higher than that of the HER2+EpCam antibody combination.

The present invention uses CTC single cell sequencing data of breast cancer patients to accurately characterize the gene expression characteristics of breast cancer CTC cells. By comparing the gene expression abundance of single cells in normal human blood, it screens breast cancer CTC-specific expression genes and integrates CTC RNA-seq data. and protein expression profiles to further screen out a series of potential surface markers of CTCs in breast cancer blood (FAIM2, KCNJ4, GABRD and UPK1B). Microfluidic chips coated with surface marker antibodies were used to detect the authenticity and effectiveness of specific surface markers for breast cancer CTCs. On this basis, we further optimized and screened out the antibody combination HER2, EpCam, FAIM2 and GABRD and the combination of HER2, EpCam, KCNJ4 and GABRD with higher CTC capture efficiency. CTC capture test experiments were conducted in breast cancer cell lines and breast cancer patient blood samples. The results showed that the optimized antibody combination was significantly better than the current mainstream antibody capture scheme in terms of capture efficiency.

The breast cancer CTC-specific antibodies and combinations provided by the present invention solve the problems of low CTC detection rate and low purity in the blood of breast cancer patients. The novel antibody combination identified in the present invention can significantly improve the enrichment and capture efficiency of CTCs in breast cancer patients, which is helpful for real-time monitoring of tumor dynamics and evaluation of therapeutic effects in breast cancer patients. Effectively promoting the realization of real-time precise individual treatment, it has broad application prospects and can create economic benefits while improving social benefits.

Claims (3)

1. The application of an antibody corresponding to a cell surface marker in the preparation of a reagent for detecting breast cancer, which is characterized in that the antibody corresponding to the cell surface marker is used for immune capture of breast cancer circulating tumor cells CTC; the cell surface marker includes TSPAN1 , FAIM2, KCNJ4, GABRD, PKD1L2, OR10K1, UPK1B and OR51M1; HER2 antibody, EpCam antibody, FAIM2 antibody and GABRD antibody are used in combination, or HER2 antibody, EpCam antibody, KCNJ4 antibody and GABRD antibody are used in combination. 2. The application of an antibody corresponding to a cell surface marker in preparing a reagent for detecting breast cancer according to claim 1, wherein the process of immune capture of breast cancer circulating tumor cells CTC is as follows: Step 1: Coat the microfluidic chip with cell surface marker antibodies; Step 2: Flow the cultured breast cancer cell suspension or breast cancer patient blood sample through the chip at a certain speed; Step 3: Use the collection solution to collect the cells captured by the chip and count them; the count represents the ability of the antibody to capture breast cancer cells. 3. Application of an antibody corresponding to a cell surface marker in preparing a reagent for detecting breast cancer according to claim 1, characterized in that the screening method for cell surface markers includes the following steps: according to breast cancer in a public database RNA-seq and scRNA-seq sequencing data of circulating tumor cell CTC samples of patients, analyze the specific expression profile information and heterogeneity characteristics of CTCs in breast cancer patients, and obtain CTC highly expressed genes; based on the gene expression abundance of other cell types in blood samples The information is filtered and combined with the surface protein expression characteristic information to obtain CTC-specific surface markers; the obtained CTC-specific surface markers are compared with the expression profiles of breast cancer patient tumor tissue samples in the public database to determine the cell surface markers of CTCs.