Surface markers of circulating tumor cells in pancreatic cancer and their applications
By performing single-cell sequencing and big data analysis on pancreatic cancer CTCs, unique surface markers were screened out and antibody combinations were optimized, solving the problems of low efficiency and high false negative rate in existing CTC detection methods, and achieving efficient capture and detection of pancreatic cancer CTCs.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CHENGDU PRECISOME BIOTECH CO LTD
- Filing Date
- 2023-03-07
- Publication Date
- 2026-07-17
AI Technical Summary
Existing CTC detection methods suffer from low capture and enrichment efficiency and high false negative rate in pancreatic cancer patients. In particular, the differences in CTC surface markers for different types of tumors lead to poor detection efficiency.
By performing single-cell sequencing and big data analysis on CTCs from pancreatic cancer patients, a series of unique surface markers, including MFSD2B, ITGA2, PDE3A, PTPRN, OXTR, ADRA2A, BAMBI, and SLC24A3, were screened out. Antibody combinations were optimized, and antibodies using these markers were used for immunoenrichment screening to improve capture efficiency.
It significantly improved the enrichment and detection rate of CTCs in the blood of pancreatic cancer patients, reduced the false negative rate, and provided more accurate support for the monitoring and treatment of pancreatic cancer patients.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to the field of tumor diagnosis and treatment, specifically to a surface marker of circulating tumor cells in pancreatic cancer and its application. Background Technology
[0002] Circulating tumor cells (CTCs) are a collective term for various types of tumor cells present in peripheral blood. Due to spontaneous shedding from solid tumor lesions (primary and metastatic sites) during diagnosis or treatment, most CTCs undergo apoptosis or are phagocytosed after entering the peripheral blood. A small number escape and anchor, developing into metastatic lesions, increasing the risk of death for patients with malignant tumors. CTC detection captures and detects trace amounts of CTCs in peripheral blood, monitoring trends in CTC type and quantity changes to enable real-time monitoring of tumor dynamics, assessment of treatment effectiveness, and realization of real-time personalized treatment.
[0003] CTC testing is applicable to solid tumors that metastasize through the bloodstream, including nasopharyngeal carcinoma, esophageal cancer, lung cancer, breast cancer, liver cancer, bile duct cancer, gastric cancer, pancreatic cancer, colorectal cancer, kidney cancer, bladder cancer, ovarian cancer, prostate cancer, cervical cancer, endometrial cancer, and osteosarcoma. CTC testing was included in breast cancer screening guidelines in 2019.
[0004] Existing methods for detecting circulating tumor cells (CTCs) include physical methods and immunoassays. Physical methods primarily utilize microfluidics principles to separate circulating tumor cells (TCs) on a microfluidic chip, taking advantage of cell size (generally, tumor cells are larger than blood cells). However, because circulating tumor cells are not uniform in size, have a certain degree of flexibility, and have a low proportion of tumor cells, physical methods lack specificity for separation.
[0005] With in-depth research into CTC detection, immune screening has gained increasing importance, offering unparalleled efficiency compared to other methods. Immune screening techniques for CTCs include positive and negative screening. Negative screening involves using antibodies targeting surface-specific antigens to capture and remove leukocytes; theoretically, the remaining uncaptured cells are CTCs. The advantages of this method are: mature and readily available antibodies, and high efficiency in capturing leukocytes. The disadvantage is that antibodies targeting leukocytes can non-specifically bind to some poorly functioning CTCs, thus reducing the detection efficiency.
[0006] Positive screening, also known as immunoenrichment, involves using antibodies against specific tumor cell surface markers derived from epithelial cells to capture tumor cells (CTCs) via immune capture, thus achieving detection. The advantage of this method is that it is not sensitive to the state of the CTCs; therefore, as long as the CTCs express the capture marker, they can be detected. The disadvantage is that different types of tumors have different surface markers for CTCs, so using a relatively single marker for enrichment and screening inevitably leads to some missed detections. For example, currently, the CTC surface markers used in immunoenrichment methods are mainly adhesion molecules like EpCam and cytokeratin. This fixed combination of antibodies is clearly not applicable to all types of tumors. Therefore, identifying specific markers on the surface of CTCs in specific tumors and using these markers in combination will greatly improve the efficiency of enrichment, screening, and detection of CTCs in the blood of cancer patients. Summary of the Invention
[0007] To address the shortcomings of existing CTC enrichment methods and the limited types of enrichment biomarkers, this invention performs single-cell sequencing on CTCs and WBCs from pancreatic cancer patients and uses bioinformatics technology to conduct big data analysis of the sequencing results, summarizing a series of novel biomarkers unique to the CTC surface. Furthermore, by comparing the sequencing results with the protein expression profiles of a large number of pancreatic cancer patients and the population sequencing results of CTC expression profiles, common CTC-specific biomarkers are identified. Based on this, enrichment (capture) tests are performed on pancreatic cancer cell lines and blood from pancreatic cancer patients using these novel biomarkers. Surface biomarkers with enrichment capabilities are selected, and the optimal combination of new capture antibodies is optimized for immunoenrichment screening. The antibodies or antibody combinations of the novel surface biomarkers screened through the above method significantly improve the capture and enrichment efficiency of CTCs, providing more accurate support for monitoring tumor dynamics, evaluating treatment effects, and realizing real-time personalized treatment, possessing significant market value and application prospects.
[0008] In one aspect, the present invention provides a product for detecting or capturing pancreatic cancer circulating tumor cells (CTCs) in a sample, wherein the product is used to detect the level of cell surface marker gene expression products in the sample, the cell surface markers being selected from one or more of MFSD2B, ITGA2, PDE3A, PTPRN, OXTR, ADRA2A, BAMBI, and SLC24A.
[0009] In one aspect, the present invention provides the use of a product for detecting the level of cell surface marker gene expression products in a sample in the preparation of reagents, kits, or arrays for detecting or capturing pancreatic cancer circulating tumor cells (CTCs) in a sample, wherein the cell surface marker is selected from one or more of MFSD2B, ITGA2, PDE3A, PTPRN, OXTR, ADRA2A, BAMBI, and SLC24A, wherein:
[0010] The 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 products is higher than the reference level, pancreatic cancer circulating tumor cells (CTCs) are identified; or
[0011] The product is used to capture circulating tumor cells (CTCs) of pancreatic cancer in samples.
[0012] In one aspect, the present invention provides a cell surface marker expression product for diagnosing pancreatic cancer in a subject or determining the risk of developing pancreatic cancer in a subject, wherein the cell surface marker is selected from one or more of MFSD2B, ITGA2, PDE3A, PTPRN, OXTR, ADRA2A, BAMBI, and SLC24A.
[0013] In one aspect, the present invention provides the use of a product for detecting the level of gene expression products of cell surface markers in a sample in the preparation of reagents, kits or arrays for diagnosing pancreatic cancer in a subject or determining the risk of developing pancreatic cancer in a subject, wherein the cell surface markers are selected from one or more of MFSD2B, ITGA2, PDE3A, PTPRN, OXTR, ADRA2A, BAMBI and SLC24A.
[0014] In one aspect, the present invention provides a method for capturing pancreatic cancer circulating tumor cells (CTCs) from a sample, the method comprising: contacting the sample with a product targeting cell surface markers on pancreatic cancer CTCs to capture the circulating tumor cells, wherein the cell surface markers are selected from one or more of MFSD2B, ITGA2, PDE3A, PTPRN, OXTR, ADRA2A, BAMBI, and SLC24A, and the product is immobilized on the surface of a solid support.
[0015] In one aspect, the present invention provides an apparatus for capturing pancreatic cancer circulating tumor cells (CTCs) from a sample, the apparatus comprising a channel having a cell-capturing surface, wherein a product targeting cell surface markers on pancreatic cancer circulating tumor cells (CTCs) is immobilized to the cell-capturing surface.
[0016] In one aspect, the present invention provides a kit or array for detecting or capturing pancreatic cancer circulating tumor cells (CTCs) in a sample, wherein the kit or array comprises the product described above.
[0017] In one aspect, the present invention provides a kit or array for assisting in the diagnosis of pancreatic cancer in a subject or determining the risk of developing pancreatic cancer in a subject, wherein the kit or array comprises the product described above. Attached Figure Description
[0018] Figure 1 The screening of circulating tumor cell surface marker genes in pancreatic cancer is illustrated. Among them, Figure 1 A shows the gene expression abundance distribution of circulating tumor cell surface markers in pancreatic cancer; Figure 1 B shows the specificity of expression levels of circulating tumor cell surface marker proteins in pancreatic cancer.
[0019] Figure 2 shows the preliminary screening of circulating tumor cell surface markers: MFSD2B, ITGA2, PDE3A, PTPRN, OXTR, ADRA2A, BAMBI, and SLC24A3, and the segmental distribution of these proteins on the cell membrane structure. Figure 2A The distribution of MFSD2B, PDE3A, OXTR, and BAMBI in the cell membrane structure is shown. Figure 2B The segmental distribution of ITGA2, PTPRN, ADRA2A, and SLC24A3 on the cell membrane structure is shown.
[0020] Figure 3 The diagram illustrates the sample capture, collection, and counting apparatus and workflow. Peripheral venous whole blood from pancreatic cancer patients flows through a microfluidic chip immobilized with EpCam, CA199, and novel biomarker antibodies. Tumor cells are enriched on the chip, and the enriched cells are then counted using fluorescent staining. Specifically, Figure 3 A is the capture and collection device. Figure 3 B represents a fluorescence scanning counting system. Figure 3 C is a schematic diagram of the sample enrichment, separation, and counting process.
[0021] Figure 4 The efficiency of enriching and screening pancreatic cancer cells SU.86.86 is shown. SU.86.86 cells were passed through microfluidic chips immobilized with antibodies against ITGA2, PTPRN, OXTR, BAMBI, MFSD2B, ADRA2A, PDE3A, and SLC24A3, respectively. The capture and enrichment efficiency of each chip for SU.86.86 cells is shown. The capture and enrichment efficiencies, from highest to lowest, are: ITGA2, PTPRN, OXTR, BAMBI, MFSD2B, ADRA2A, PDE3A, and SLC24A3.
[0022] Figure 5The diagram shows the capture and enrichment efficiency of SU.86.86 cells through microfluidic chips immobilized with EpCam antibody in combination with MFSD2B, ITGA2, PDE3A, PTPRN, OXTR, ADRA2A, BAMBI, or SLC24A3 antibodies. The capture efficiency of EpCam antibody combined with ITGA2, PTPRN, OXTR, or BAMBI antibodies was significantly higher than that of EpCam antibody alone.
[0023] Figure 6 The diagram shows the capture and enrichment efficiency of SU.86.86 cells by passing through microfluidic chips immobilized with CA199 antibody in combination with MFSD2B, ITGA2, PDE3A, PTPRN, OXTR, ADRA2A, BAMBI, or SLC24A3 antibodies. The capture efficiency of CA199 antibody combined with ITGA2, PTPRN, OXTR, or BAMBI antibodies was significantly higher than that of CA199 antibody alone.
[0024] Figure 7 The diagram illustrates the capture and enrichment efficiency of SU.86.86 cells through microfluidic chips immobilized with either CA199 or EpCam antibody combinations, along with antibodies against MFSD2B, ITGA2, PDE3A, PTPRN, OXTR, ADRA2A, BAMBI, or SLC24A3. The combination of CA199 and EpCam antibodies with ITGA2, PTPRN, OXTR, or BAMBI showed significantly higher capture efficiency than the combination of CA199 and EpCam alone.
[0025] Figure 8 The results showed that SU.86.86 cells were captured more efficiently by passing through combinations of CA199 and EpCam antibodies combined with ITGA2 and PTPRN antibodies, ITGA2 and OXTR antibodies, ITGA2 and BAMBI antibodies, PTPRN and OXTR antibodies, PTPRN and BAMBI antibodies, or OXTR and BAMBI antibodies, respectively. These results were significantly higher than the capture efficiency of the CA199 and EpCam antibody combination.
[0026] Figure 9This study demonstrates the use of combined antibodies against CA199, EpCam, ITGA2, and PTPRN, as well as combined antibodies against CA199, EpCam, OXTR, and PTPRN, to coat microarrays. Four types of pancreatic cancer cells (Capan-1, CFPAC-1, Hs 766T, and SU.86.86) were cultured, with 1000 cells of each type counted. These 1000 cells were then flowed through the microarray at a controlled flow rate, and the captured cells were collected using a collection solution. The capture efficiency of the combined antibodies against the four types of pancreatic cancer cells was counted and calculated. The combination of CA199 and EpCam antibodies with ITGA2 and PTPRN antibodies, or the combination of PTPRN and OXTR antibodies, showed high capture and enrichment efficiencies for the four types of pancreatic cancer cells, exceeding the capture efficiency of the CA199 and EpCam antibody combination alone.
[0027] Figure 10 The study demonstrated that the combination of CA199 and EpCam antibodies, in combination with the combination of ITGA2 and PTPRN antibodies, achieved a significantly higher capture and enrichment efficiency for four types of pancreatic cancer cells mixed in the blood than the combination of CA199 and EpCam antibodies alone.
[0028] Figure 11 The study demonstrated the capture and enrichment of CTCs in blood samples from 10 pancreatic cancer patients using a combination of CA199 and EpCam antibodies in combination with ITGA2 and PTPRN antibodies. The overall number of CTCs captured and enriched by the combination of CA199 and EpCam antibodies in combination with ITGA2 and PTPRN antibodies was higher than that captured and enriched by the combination of CA199 and EpCam antibodies alone. Detailed Implementation
[0029] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.
[0030] In this document, the terms “individual,” “patient,” or “subject” are used interchangeably and refer to any single animal, preferably a mammal (including non-human animals such as, for example, cats, dogs, horses, rabbits, zoo animals, cattle, pigs, sheep, and non-human primates), that is desired to be treated. In a particular embodiment, the patient in this document is a human being. A patient can be a “cancer patient,” that is, a patient who has cancer, is at risk of developing cancer, or has one or more symptoms of cancer.
[0031] In this article, the term "tumor" refers to all proliferative cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.
[0032] In this article, the terms “cancer” and “cancerous” refer to or describe a physiological disorder in mammals that is typically characterized by unregulated cell growth.
[0033] In this article, the terms “cancer,” “cancerous,” “proliferative disorder,” “proliferative disease,” and “tumor” are used without mutual exclusion.
[0034] In this article, the term "symptom" refers to any condition that would benefit from treatment, including but not limited to chronic and acute symptoms or diseases, including those pathological conditions that make mammals susceptible to the symptoms discussed.
[0035] In this document, the term "tumor cell" refers to any tumor cell present in a tumor or its sample. 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.
[0036] In this document, the term "circulating tumor cells" or "CTCs" refers to tumor cells that have detached from a tumor and are present in the blood (i.e., in circulation). Cellular markers (e.g., marker genes) that can be used to identify and / or isolate CTCs from other components of the blood are described below. In some embodiments, CTCs may be pancreatic cancer CTCs.
[0037] In this article, the term “cell surface” is used according to its normal meaning in this field, and therefore includes the cell exterior that can be accessed by binding to proteins and other molecules.
[0038] In this document, the term "tumor cell surface marker" refers to biomolecules such as proteins, carbohydrates, and glycoproteins. These biomolecules are specifically, preferably, or differentially expressed on tumor cells and / or are found to be associated with tumor cells, thus serving as preferred or specific targets for tumors. In specific embodiments, dominant expression may be dominant expression compared to other cells in the organism, or dominant expression in a specific region of the organism (e.g., in a specific organ or tissue).
[0039] In this document, the term "sample" or "test sample" refers to a sample obtained or isolated 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. The term also includes mixtures of the above samples. The term "test sample" also includes untreated or pretreated (or pre-processed) biological samples. In some embodiments, a test sample may include cells from a subject. In some embodiments, a test sample may be a tumor cell test sample; for example, the sample may include cancer cells, cells from a tumor, and / or tumor biopsies. In some embodiments, a test sample may be a blood sample. A test sample can be obtained by removing a cell sample from a subject, but it can also be done by using previously isolated cells (e.g., isolated at a previous time point and isolated by the same person or another person). Furthermore, a test sample may be a freshly collected or previously collected sample.
[0040] In this document, the term "antigen" refers to a substance containing an epitope, against which an immune response is directed and / or generated. Preferably, in the context of this invention, an antigen is a molecule that, optionally after processing, induces an immune response, preferably antigen-specific. The term "antigen" particularly includes proteins, peptides, polysaccharides, nucleic acids, especially RNA and DNA, and nucleotides.
[0041] In this document, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds, and includes any molecule containing its antigen-binding moiety. The term "antibody" includes monoclonal antibodies and antibody fragments or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single-chain antibodies such as scFv antibody fragments, and antigen-binding antibody fragments such as Fab and Fab' fragments, as well as all recombinant forms of antibodies, such as antibodies expressed in prokaryotes, non-glycosylated antibodies, and any antigen-binding antibody fragments and derivatives described herein. Each heavy chain consists of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which intersperse with more conserved regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The constant regions of the antibody mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.
[0042] In this article, the term "detection" includes any detection method, including direct and indirect detection.
[0043] In one aspect, the present invention provides a product for detecting or capturing pancreatic cancer circulating tumor cells (CTCs) in a sample, wherein the product is used to detect the level of cell surface marker gene expression products in the sample, the cell surface markers being selected from one or more of MFSD2B, ITGA2, PDE3A, PTPRN, OXTR, ADRA2A, BAMBI, and SLC24A.
[0044] In one aspect, the present invention provides the use of a product for detecting the level of cell surface marker gene expression products in a sample in the preparation of reagents, kits, or arrays for detecting or capturing pancreatic cancer circulating tumor cells (CTCs) in a sample, wherein the cell surface marker is selected from one or more of MFSD2B, ITGA2, PDE3A, PTPRN, OXTR, ADRA2A, BAMBI, and SLC24A, wherein:
[0045] The 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 products is higher than the reference level, pancreatic cancer circulating tumor cells (CTCs) are identified; or
[0046] The product is used to capture circulating tumor cells (CTCs) of pancreatic cancer in samples.
[0047] In one aspect, the present invention provides a cell surface marker expression product for diagnosing pancreatic cancer in a subject or determining the risk of developing pancreatic cancer in a subject, wherein the cell surface marker is selected from one or more of MFSD2B, ITGA2, PDE3A, PTPRN, OXTR, ADRA2A, BAMBI, and SLC24A.
[0048] In one aspect, the present invention provides the use of a product for detecting the level of gene expression products of cell surface markers in a sample in the preparation of reagents, kits or arrays for diagnosing pancreatic cancer in a subject or determining the risk of developing pancreatic cancer in a subject, wherein the cell surface markers are selected from one or more of MFSD2B, ITGA2, PDE3A, PTPRN, OXTR, ADRA2A, BAMBI and SLC24A.
[0049] In one aspect, the present invention provides a method for capturing pancreatic cancer circulating tumor cells (CTCs) from a sample, the method comprising: contacting the sample with a product targeting cell surface markers on pancreatic cancer CTCs to capture the circulating tumor cells, wherein the cell surface markers are selected from one or more of MFSD2B, ITGA2, PDE3A, PTPRN, OXTR, ADRA2A, BAMBI, and SLC24A, and the product is immobilized on the surface of a solid support.
[0050] In one aspect, the present invention provides an apparatus for capturing pancreatic cancer circulating tumor cells (CTCs) from a sample, the apparatus comprising a channel having a cell-capturing surface, wherein a product targeting cell surface markers on pancreatic cancer circulating tumor cells (CTCs) is immobilized to the cell-capturing surface.
[0051] In some embodiments, the device is a microfluidic device.
[0052] In some embodiments, the expression product is a nucleic acid.
[0053] In some implementations, the product is used to determine the level of the expression product using methods selected from the group consisting of: RT-PCR; Northern blotting; microarray-based expression analysis; next-generation sequencing; and RNA in situ hybridization.
[0054] In some implementations, quantitative RT-PCR is used to determine the level of the expression product.
[0055] In some embodiments, the expression product is a polypeptide.
[0056] In some embodiments, the product is used to determine the level of the expression product using a method selected from the group consisting of: Western blotting; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunoassay; sandwich assay; fluorescence in situ hybridization (FISH); immunohistochemical staining; immunofluorescence assay; mass spectrometry; FACS; and immunoelectrophoresis.
[0057] In some embodiments, the cell surface markers also include CA199 and / or EpCam.
[0058] In some embodiments, the cell surface marker comprises a combination of cell surface markers selected from any of the following:
[0059] (1) EpCam, and any one of the cell surface markers selected from ITGA2, PTPRN, OXTR and BAMBI;
[0060] (2) CA199, and any one of the cell surface markers selected from ITGA2, PTPRN, OXTR and BAMBI;
[0061] (3) CA199 and EpCam, and any one of the cell surface markers selected from ITGA2, PTPRN, OXTR and BAMBI;
[0062] (4) CA199 and EpCam, and any combination selected from the combination of ITGA2 and PTPRN, ITGA2 and OXTR, ITGA2 and BAMBI, PTPRN and OXTR, PTPRN and BAMBI, and OXTR and BAMBI.
[0063] In some specific embodiments, the cell surface markers comprise a combination of CA199, EpCam, ITGA2, and PTPRN, or a combination of CA199, EpCam, OXTR, and PTPRN.
[0064] In some embodiments, the product is an antibody against a cell surface marker.
[0065] In some specific embodiments, the antibody is a monoclonal antibody.
[0066] In one aspect, the present invention provides a kit or array for detecting or capturing pancreatic cancer circulating tumor cells (CTCs) in a sample, wherein the kit or array comprises the aforementioned products.
[0067] In one aspect, the present invention provides a kit or array for assisting in the diagnosis of pancreatic cancer in a subject or determining the risk of developing pancreatic cancer in a subject, wherein the kit or array comprises the aforementioned products.
[0068] This invention addresses the shortcomings of existing CTC capture products by performing single-cell sequencing on CTC sample data isolated from pancreatic cancer patients and comparing it with pancreatic cancer data in public databases to screen a series of potential CTC surface markers in the blood of pancreatic cancer patients. Furthermore, through bioinformatics analysis, candidate circulating tumor cell surface markers for pancreatic cancer (ITGA2, PTPRN, OXTR, BAMBI, MFSD2B, ADRA2A, PDE3A, and SLC24A3) were selected. These markers, individually or in combination with widely used markers (EpCam and CA199), were used in microfluidic chips coated with surface marker antibodies to capture and enrich CTCs in pancreatic cancer cell lines and blood samples from pancreatic cancer patients in vitro. The authenticity and effectiveness of the surface markers were analyzed. Antibody combinations with high CTC capture efficiency were optimized and screened (i.e., CA199+EpCam+ITGA2+PTPRN, and CA199+EpCam+OXTR+PTPRN). Compared to existing CTC enrichment and screening products, this invention significantly improves the enrichment quantity of CTC cells in the blood of pancreatic cancer patients and the detection rate of CTCs in patient blood samples by using antibodies or antibody combinations targeting novel surface markers. This greatly reduces the false negative rate and solves the problems of low detection rate and small quantity of CTCs in the blood of pancreatic cancer patients in the past. It greatly improves the capture and enrichment efficiency of CTCs, providing strong support for more pancreatic cancer patients to monitor tumor dynamics and evaluate treatment effects, and enabling doctors to achieve real-time, precise, and individualized treatment for patients. It has huge market value and application prospects, benefiting more cancer patients.
[0069] The present invention will now be described in more detail with reference to specific embodiments. However, these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.
[0070] Example
[0071] Example 1: Screening and determination of circulating tumor cell surface markers
[0072] 1) Screening and analysis of circulating tumor cell surface marker genes
[0073] Circulating tumor cells (CTCs) from the blood of pancreatic cancer patients were collected using flow cytometry and single-cell sequencing was performed. Published single-cell sequencing data from blood samples of pancreatic cancer patients were also searched and compared. Using analytical tools such as Cellranger and Seurat, expression profiles of different cell types in circulating tumor cells and blood samples from pancreatic cancer patients were obtained. Differences in gene expression between circulating tumor cells and other cell types in the blood were compared to analyze the specific expression profiles and heterogeneity characteristics of circulating tumor cells from pancreatic cancer patients, and to identify genes encoding surface markers specific to circulating tumor cells. Based on this, the genes encoding these unique circulating tumor cell surface markers were compared with tumor protein expression profiles and circulating tumor cell population sequencing expression profiles from pancreatic cancer patients in public databases to identify common surface marker genes: MFSD2B, ITGA2, PDE3A, PTPRN, OXTR, ADRA2A, BAMBI, and SLC24A3. Figure 1 A and Figure 1 B). The comparison of expression levels of the screened surface marker genes in various cell types showed that their expression level in CTCs was much higher than that in other cell types.
[0074] 2) Perform protein sequence analysis on the genes analyzed in step 1) above.
[0075] Biomarkers with relatively high expression levels of both transmembrane and extracellular regions were screened from the above-mentioned markers for testing. The preliminary screening of circulating tumor cell surface markers included: MFSD2B, ITGA2, PDE3A, PTPRN, OXTR, ADRA2A, BAMBI, and SLC24A3. Figure 2 shows the distribution of these proteins on the cell membrane structure in the preliminary screening of circulating tumor cell surface markers: MFSD2B, ITGA2, PDE3A, PTPRN, OXTR, ADRA2A, BAMBI, and SLC24A3. Figure 2A and Figure 2B ).
[0076] 3) Capture and enrich pancreatic cancer cells using antibodies corresponding to surface markers.
[0077] First, the collected peripheral blood is separated, and the separated cells are slowly pumped through a microfluidic chip coated with a combination of surface marker antibodies. Cells with surface markers are captured and enriched by the chip, then the captured cells are eluted and collected, stained with fluorescent staining reagents, and finally scanned and counted using a fluorescence counter.
[0078] Antibodies against the markers selected in step 2) above were used to perform enrichment and screening experiments on relevant tumor cells, including pancreatic cancer cell lines and blood samples from pancreatic cancer patients. The experiments verified the enrichment and screening capabilities of the aforementioned surface markers for pancreatic cancer tumor cells and CTCs in the blood.
[0079] The results showed that the enrichment and screening ability of the chip coated with the combined antibody of CA199, EpCam, OXTR, and PTPRN for CTCs in pancreatic cancer cell lines and blood samples from pancreatic cancer patients was significantly higher than that of CA199 and EpCam single antibodies. The device of the product is as follows... Figure 3 As shown, it mainly consists of two parts: first, a capture and collection device, which includes a microfluidic injection pump, a combination of antibodies coated with different surface markers, and a collection device; second, a fluorescence scanning and counting system, which can scan the cells in the sample loading chamber and then count them based on different fluorescence colors.
[0080] 4) Validate the enrichment and screening capabilities of candidate biomarkers using the pancreatic cancer cell line SU.86.86.
[0081] Prepare or purchase antibodies against MFSD2B, ITGA2, PDE3A, PTPRN, OXTR, ADRA2A, BAMBI, and SLC24A3, including: MFSD2B antibody (catalog number: orb185620, manufacturer: Biorbyt); ITGA2 antibody (catalog number: A72031-100, manufacturer: Epigentek); PDE3A antibody (catalog number: NP_000912, manufacturer: United States Biological); and PTPRN antibody (catalog number: PA5-64913). The following antibodies were used: Thermoma (Catalog No.: O8251-04C-100ug, Manufacturer: United States Biological); LifeSpan BioSciences (Catalog No.: LS-B7669-50); Novus Biologicals (Catalog No.: NBP2-58990); and Affinity Biosciences (Catalog No.: DF4524). The above antibodies were used individually or in combination to coat the microarray, and the enrichment and screening ability of each antibody for pancreatic cancer cells was tested.
[0082] In this experiment, classic pancreatic cancer cells SU.86.86 were selected as the experimental cells. After coating the microarray with antibodies, the cultured SU.86.86 cells were counted, with 1000 cells per group, flowing through the microarray at a certain speed. The cells captured by the microarray were collected using a collection solution and counted. The capture ability of different antibodies against SU.86.86 pancreatic cancer cells was counted.
[0083] First, the ability of any one of the above markers to capture and enrich pancreatic cancer tumor cells was tested using antibodies alone (experimental results are shown in (a)). Then, the above marker antibodies were combined with EpCam and / or CA199 antibodies separately or in an overlapping manner to test the ability of the superimposed antibodies to capture and enrich pancreatic cancer tumor cells (experimental results are shown in (b)-(e)).
[0084] The experimental results are as follows:
[0085] (a) Percentage of cells enriched and captured by a single antibody
[0086] Depend on Figure 4 It can be seen that the selected markers all have a certain capture and enrichment ability for SU.86.86 cells through single antibody enrichment screening experiments. The capture and enrichment efficiency from high to low is ITGA2, PTPRN, OXTR, BAMBI, MFSD2B, ADRA2A, PDE3A and SLC24A3.
[0087] (b) The above antibodies were combined with EpCam antibodies respectively, and the percentage of captured and enriched cells (total number of input cells) was tested.
[0088] Depend on Figure 5 It is evident that the capture efficiency of EpCam antibody combined with ITGA2, PTPRN, OXTR, or BAMBI antibodies is higher than that of EpCam antibody alone.
[0089] (c) The above antibodies were combined with CA199 antibody respectively, and the percentage of captured and enriched cells was tested (total number of cells input).
[0090] Depend on Figure 6 It is evident that the capture efficiency of CA199 antibody combined with ITGA2, PTPRN, OXTR, or BAMBI antibodies is higher than that of CA199 antibody alone.
[0091] (d) The above antibodies were combined with CA199 and EpCam antibodies, respectively, and the percentage of pancreatic cancer cells captured and enriched by the antibody superposition was tested (total number of cells input).
[0092] Depend on Figure 7 It is evident that the combination of CA199 and EpCam antibodies with ITGA2, PTPRN, OXTR, or BAMBI antibodies exhibits higher capture efficiency, significantly exceeding that of the combination of CA199 and EpCam antibodies alone.
[0093] (e) The CA199 and EpCam antibodies were combined in pairs with four selected antibodies to test the percentage of pancreatic cancer cells captured and enriched by the combined antibodies (total number of cells entered).
[0094] Depend on Figure 8 It is evident that the combination of CA199 and EpCam antibodies with ITGA2 and PTPRN antibodies, ITGA2 and OXTR antibodies, ITGA2 and BAMBI antibodies, PTPRN and OXTR antibodies, PTPRN and BAMBI antibodies, or OXTR and BAMBI antibodies exhibits higher capture efficiency, significantly higher than that of the CA199 and EpCam antibody combination.
[0095] The above experiments show that, using the pancreatic cancer cell line SU.86.86, highly efficient capture antibodies were selected: ITGA2, PTPRN, OXTR, BAMBI, MFSD2B, ADRA2A, PDE3A, and SLC24A3. Among them, ITGA2, PTPRN, OXTR, and BAMBI antibodies showed the highest capture efficiency. Combining the EpCam and CA199 antibody with ITGA2, PTPRN, OXTR, and BAMBI individually or in combination with each of these four antibodies resulted in significantly higher capture efficiencies than existing EpCam and CA199 single or combined capture regimens. The CA199, EpCam, ITGA2, and PTPRN antibody combination and the CA199, EpCam, OXTR, and PTPRN antibody combination are the two most efficient combination regimens, achieving capture efficiencies of 98% and 95%, respectively.
[0096] Example 2: Capture efficiency of combined antibodies against different pancreatic cancer cells
[0097] The chips were coated with a combination antibody of CA199, EpCam, ITGA2, and PTPRN, as well as another combination antibody of CA199, EpCam, OXTR, and PTPRN. Four types of pancreatic cancer cells, Capan-1, CFPAC-1, Hs 766T, and SU.86.86, were cultured, with 1000 cells of each type. The 1000 cells of each type of pancreatic cancer cell were added to 7.5 ml of sample loading solution and mixed. An automated sample pump was used to pump the sample into the chip at a certain flow rate. The cells captured and enriched by the chip were collected, stained with fluorescence, and scanned and counted using a fluorescence scanner to calculate the capture ability of the combination antibody against the four types of pancreatic cancer cells.
[0098] The results showed that the combination of CA199 and EpCam antibodies, combined with the combination of ITGA2 and PTPRN antibodies, or the combination of PTPRN and OXTR antibodies, had high capture and enrichment efficiency for four types of pancreatic cancer cells, all of which were higher than the capture efficiency of the combination of CA199 and EpCam antibodies.
[0099] Example 3: Capture efficiency of combined antibodies against different pancreatic cancer cells in the blood
[0100] As shown in Example 2, the combination of four antibodies—CA199, EpCam, ITGA2, and PTPRN—exhibited superior capture and enrichment capabilities against several typical pancreatic cancer cells compared to the combination of CA199, EpCam, OXTR, and PTPRN. Therefore, in this example, only the combination of CA199, EpCam, ITGA2, and PTPRN antibodies was used to coat the microarray. Four types of cultured pancreatic cancer cells—Capan-1, CFPAC-1, Hs 766T, and SU.86.86—were counted at 1000 cells each. 1000 cells of each of the four types of pancreatic cancer cells were added to 7.5 ml of whole blood from healthy individuals, mixed, and treated with erythrocyte lysis buffer. The collected cell pellet was then added to 7.5 ml of protective buffer to form a cell suspension. An automated sample pump was used to pump the cell suspension into the microarray at a specific flow rate. The captured and enriched cells were collected, stained with fluorescence, and scanned and counted using a fluorescence scanner. The capture capability of the combined antibodies against the four types of pancreatic cancer cells in whole blood was calculated.
[0101] Cells captured by the chip are collected with a collection solution at a controlled flow rate and then stained with a fluorescent marker. After scanning and counting, the chip's ability to capture four types of pancreatic cancer cells from whole blood is calculated.
[0102] The results showed that the combination of CA199 and EpCam antibodies, combined with the combination of ITGA2 and PTPRN antibodies, had a significantly higher capture and enrichment efficiency for four types of pancreatic cancer cells mixed in the blood than the combination of CA199 and EpCam antibodies alone.
[0103] Example 4: Capture ability of combined antibodies against CTCs in the blood of pancreatic cancer patients
[0104] A microarray was coated with a combination of four antibodies: CA199, EpCam, ITGA2, and PTPRN. 7.5 ml of whole blood was drawn from pancreatic cancer patients and treated with erythrocyte lysis buffer. The collected cell pellet was then mixed with 7.5 ml of preservative buffer to form a cell suspension. An automated sample pump was used to pump the cell suspension into the microarray at a controlled flow rate. Cells captured by the microarray were collected using a collection buffer and stained with a fluorescent marker. A fluorescence scanning counter was used to analyze the sample and record the number of centrally acting tumor cells (CTCs) captured by the antibody-coated microarray in the blood of pancreatic cancer patients.
[0105] The results showed that when the combination of CA199 and EpCam antibodies, along with the combination of ITGA2 and PTPRN antibodies, was used to capture and enrich CTCs in blood samples from 10 pancreatic cancer patients, the overall number of CTCs captured and enriched by the combination of CA199 and EpCam antibodies, along with the combination of ITGA2 and PTPRN antibodies, was higher than the number of CTCs captured and enriched by the combination of CA199 and EpCam antibodies alone.
Claims
1. Use of a product for detecting the level of a cell surface marker gene expression product in a sample in the preparation of a reagent, kit, or array for detecting or capturing pancreatic cancer circulating tumor cells (CTCs) in a sample, wherein the cell surface marker is PTPRN; and the product is an antibody against the cell surface marker.
2. The use as described in claim 1, wherein, The cell surface markers also include CA199 and / or EpCam.
3. The use as described in claim 1, wherein, The cell surface markers comprise a combination of cell surface markers selected from any one of the following: (1) EpCam and PTPRN; (2) CA199 and PTPRN; (3) CA199, EpCam, and PTPRN; (4) CA199 and EpCam, and any combination selected from the combination of ITGA2 and PTPRN, the combination of PTPRN and OXTR, and the combination of PTPRN and BAMBI.
4. The use as described in claim 1, wherein, The cell surface markers include a combination of CA199, EpCam, ITGA2, and PTPRN, or a combination of CA199, EpCam, OXTR, and PTPRN.
5. The use as described in any one of claims 1-4, wherein, The antibody is a monoclonal antibody.