Method for screening exosome biomarkers derived from drug-loaded breast tumor stem cells

Abstract

The invention belongs to the technical field of biomedicine, and discloses a method for screening a drug-loaded breast tumor stem cell-derived exosome biomarker, which is characterized in that: quantitative proteomics is combined with biosignal analysis to screen exosome key proteins with significant difference in breast cancer; the biological function and regulation mechanism of the exosome key protein in drug resistance of breast tumor stem cells are determined through clinical level and cellular level. Aiming at the difficult problem of drug resistance specificity diagnosis and treatment of breast tumor, research is planned to be carried out in the field of tumor stem cell and exosome hotspot scientific research, and key proteins with significant difference in the breast tumor stem cell source exosome are screened through quantitative proteomics, protein expression profiles and databases; clinical sample detection and in-vivo and in-vitro experiments are combined to deeply discuss the regulation mechanism of the key protein in breast tumor drug resistance, it is clear that the key protein possibly becomes a biomarker for early diagnosis, curative effect evaluation and prognosis judgment of breast cancer, and help is better provided for precise targeted treatment of breast tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a method for screening drug-loaded exosome biomarkers derived from breast tumor stem cells. Background Art

[0002] Breast cancer is one of the most common malignancies worldwide, with morbidity and mortality rates rising rapidly year by year, becoming a serious medical and social problem. Cancer stem cells, characterized by high invasiveness, tumorigenicity, and rapid self-renewal, are the primary cause of tumor recurrence and treatment resistance. Exploring treatments for breast cancer from a cancer stem cell perspective offers the potential to improve clinical efficacy and reduce mortality. Exosomes have been a research hotspot in life sciences and translational medicine in recent years. They mediate intercellular communication and participate in the pathogenesis of various diseases. As highly efficient targeted drug delivery vehicles, they provide potential diagnostic markers and therapeutic targets for various diseases, holding broad clinical application prospects. Tumor exosomes play an important role in noninvasive disease diagnosis, liquid tumor biopsies, precision drug development, and clinical efficacy monitoring. Research on exosome molecular biomarkers is a promising approach for diagnosing, monitoring, and treating malignant tumors.

[0003] Breast cancer is the most common malignant tumor in women. Its high metastasis and mortality rates severely impact women's physical and mental health, placing a heavy burden on Chinese society and healthcare. Traditional breast cancer treatments combine surgery with chemotherapy and radiotherapy, but most patients do not experience obvious clinical symptoms in the early stages, resulting in a missed window of optimal treatment. Addressing this issue more effectively is a key focus of national and regional research and development in health. Developing more precise, clinically relevant diagnostic and therapeutic products (targeted drugs, diagnostic kits) tailored to specific diseases and their clinical translation is a strategic and forward-looking scientific priority in my country. Therefore, the search for sensitive breast cancer diagnostic markers for early clinical monitoring and the exploration of new therapeutic targets for breast cancer are of vital importance. Exosomes have been a research hotspot in recent years, playing a crucial role in mediating intercellular communication in the pathophysiology and pathology of various diseases. Capturing exosomes from body fluids and identifying their associated mRNA and protein targets as biomarkers for cancer detection has attracted widespread attention.

[0004] Through the above analysis, the problems and defects of the existing technology are as follows:

[0005] Traditional breast cancer treatment uses a combination of surgery and chemotherapy, but most patients do not have obvious clinical symptoms in the early stages, resulting in missing the optimal treatment window. Summary of the Invention

[0006] In response to the problems existing in the prior art, the present invention provides a method for screening drug-loaded exosome biomarkers derived from breast cancer stem cells.

[0007] The present invention is achieved by a method for screening drug-loaded exosome biomarkers derived from breast cancer stem cells, comprising:

[0008] Step 1, preparation of drug-loaded breast cancer stem cell exosomes;

[0009] Step 2: Quantitative proteomics screening of key exosome proteins and bioinformatics analysis;

[0010] Step 3: Study the biological functions and molecular mechanisms of key exosome proteins in breast cancer drug resistance;

[0011] Step 4: Screening and clinical translational application of drug-loaded breast cancer stem cell exosome biomarkers.

[0012] Furthermore, the drug-loaded breast cancer stem cell exosomes are prepared by:

[0013] (1) Tissue specimens from clinically diagnosed triple-negative breast cancer patients were collected, primary cells were isolated and cultured through mechanical digestion, and stem cell markers Nanog, OCT4, ALDH1, and ABCG2 were identified to confirm that the cells were derived from breast cancer stem cells.

[0014] (2) Cyclophosphamide, a conventional chemotherapy drug for triple-negative breast cancer patients, was selected and treated with a certain drug concentration in breast cancer stem cells to construct drug-resistant stem cell lines;

[0015] (3) The stem cell culture supernatants from the untreated and treated groups were collected, and exosomes derived from breast cancer stem cells were isolated and identified by density gradient ultracentrifugation;

[0016] (4) Based on the construction of drug-resistant breast cancer stem cell lines, exosomes were isolated and combined to prepare drug-loaded breast cancer stem cell exosomes.

[0017] Furthermore, the quantitative proteomics screening of key exosome proteins and bioinformatics analysis:

[0018] (1) TMT quantitative proteomics analysis screened differentially expressed proteins in exosomes. Bioinformatics analysis screened functional proteins with significant fold differences and closely related to stem cell regulation. RT-PCR preliminary verification identified key proteins that were closely related to triple-negative breast cancer resistance and were stably differentially expressed.

[0019] (2) Collect blood samples from 100 triple-negative breast cancer patients and normal controls, and use RT-PCR to further verify the universality of the expression of the above key proteins in breast cancer, and preliminarily determine whether they can be used as candidate biomarkers.

[0020] Furthermore, the biological functions and molecular mechanisms of the key exosome proteins in breast cancer drug resistance are studied:

[0021] (1) Construct key protein interference / overexpression lentiviral vectors, transfect breast cancer stem cells / triple-negative breast cancer cell lines, and detect the effects of key proteins on breast cancer stemness regulation by RT-PCR and Western blotting;

[0022] (2) Construct a breast cancer tumor-bearing animal model, inject key exosomal proteins, detect the corresponding phenotypes or the expression of downstream key molecules, and verify the biological functions of key exosomal proteins in breast cancer;

[0023] (3) Bioinformatics prediction of downstream target proteins, and CO-IP / laser confocal microscopy to verify the binding between the protein and the downstream target protein;

[0024] (4) TMT quantitative proteomics KEGG pathway screening of signaling pathways involved in regulation, and the use of pathway blockers / agonists to detect phenotypic changes and explore the molecular mechanism of key proteins in breast tumor resistance.

[0025] Furthermore, the screening and clinical transformation application of the drug-loaded breast cancer stem cell exosome biomarkers:

[0026] (1) Blood samples were collected from 100 patients with triple-negative breast cancer before and after cyclophosphamide chemotherapy. The relative expression of key exosome proteins was detected by RT-PCR and ELISA to verify that key proteins can be used as diagnostic markers for breast cancer resistance.

[0027] (2) Use the TCGA database to search for the expression of key proteins in a large sample of breast cancer tissues and their relationship with patients' overall survival, and evaluate the potential of key proteins as prognostic biomarkers for breast cancer at the clinical level;

[0028] (3) Apply databases and clinical samples combined with basic experiments to verify the functions and mechanisms of key proteins, and contact biopharmaceutical companies to try to develop new products for key proteins, such as key protein diagnostic kits, to assess the patient's disease status through kit testing, in order to achieve clinical translation;

[0029] Another object of the present invention is to provide a system for screening drug-loaded exosome biomarkers derived from breast cancer stem cells, comprising:

[0030] Preparation module for the preparation of drug-loaded breast cancer stem cell exosomes;

[0031] Analysis module, used for quantitative proteomics screening of key exosome proteins and bioinformatics analysis;

[0032] Research module for studying the biological functions and molecular mechanisms of key exosome proteins in breast cancer drug resistance;

[0033] Screening module for the screening and clinical translation of drug-loaded breast cancer stem cell exosome biomarkers.

[0034] Another object of the present invention is to provide a computer device comprising a memory and a processor, wherein the memory stores a computer program, and when the computer program is executed by the processor, the processor performs the steps of the method for screening drug-loaded breast cancer stem cell-derived exosome biomarkers.

[0035] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, which, when executed by a processor, causes the processor to perform the steps of the method for screening drug-loaded breast cancer stem cell-derived exosome biomarkers.

[0036] Another object of the present invention is to provide an information data processing terminal, which is used to implement the system for screening exosome biomarkers derived from drug-loaded breast cancer stem cells.

[0037] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0038] This paper uses quantitative proteomics combined with bioinformatics analysis to screen for key exosomal proteins with significant differences in breast cancer, and clarifies the biological functions and regulatory mechanisms of key exosomal proteins in breast cancer stem cell resistance at the clinical and cellular levels.

[0039] This invention addresses the challenges of specific diagnosis and treatment of breast tumor drug resistance. It intends to conduct research in the hot scientific research fields of tumor stem cells and exosomes. Through quantitative proteomics, protein expression profiling, and database screening, key proteins with significant differences in exosomes derived from breast tumor stem cells are screened. Combined with clinical sample testing and in vivo and in vitro experiments, the present invention further explores the regulatory mechanism of key proteins in breast tumor drug resistance, clarifying that they may become biomarkers for early diagnosis, efficacy evaluation, and prognosis of breast cancer, thereby providing better assistance for precise targeted treatment of breast tumors.

[0040] This research primarily utilizes precision medicine technologies, such as quantitative proteomics, to identify exosomal proteins as biomarkers for breast cancer, with high reliability and sensitivity for early diagnosis and risk assessment, and to develop new products for clinical translational research. By leveraging the combined strengths of diverse disciplinary resources, this research aligns with guidelines for social development and health, as well as the clinical translational application of key technologies, and is expected to contribute positively to a deeper understanding of breast cancer.

[0041] The present invention takes the clinical disease of breast tumor as the key research direction, which is in line with the funding scope of major diseases such as malignant tumors that are given priority support in the field of population health in the autonomous region's science and technology innovation and development plan. The present invention mainly uses precision medical technologies such as quantitative proteomics to screen protein targets that can be used for early clinical diagnosis and prognostic evaluation of breast tumors as novel biomarkers, and strengthen key technological breakthroughs in clinical diagnosis, treatment, and prognostic detection of breast tumors. At the same time, innovative products such as diagnostic kits with independent intellectual property rights are developed to further accelerate the transformation of innovative product results, comprehensively improve the level of scientific and technological prevention and treatment of breast tumors, and play a positive role in promoting more in-depth research on breast tumor diseases. This initiative is in line with the country's forward-looking strategic background for strengthening the clinical transformation of malignant tumors and has great social significance.

[0042] 1) Theory and approach are innovative and feasible: This research focuses on the current challenges in treating breast cancer, a major health threat to women in my country. Focusing on the applied research hotspots of cancer stem cells and exosomes, and drawing on the existing research background of breast tumor markers, this research actively analyzes the bottlenecks in treatment. Using novel proteomics techniques to analyze key exosome proteins, this research aims to develop diagnostic kits for these key proteins for clinical translational applications. This project integrates interdisciplinary disciplines such as basic medicine, clinical medicine, and experimental animal science, providing a solid theoretical foundation for its successful implementation.

[0043] (2) The project proposal has sufficient basis and feasible preliminary experimental foundation: The project applicant is mainly engaged in basic and clinical research on tumor stem cells. Currently, they have undertaken provincial, ministerial and university-level projects and participated in national scientific research projects as a key member. They have a certain preliminary scientific research foundation. In the early stage of this invention, they have successfully isolated and cultured breast tumor stem cells and obtained exosomes. They have carried out a lot of research work, which can ensure the smooth implementation of the project.

[0044] (3) Key technologies and experimental platforms are feasible: The present invention relies on the Biochip Research Center and Stem Cell Research Institute of the Ningxia Medical University General Hospital, which have a mature quality control system for the key technologies involved in the project. The Biochip Center has a complete biochip sequencing and verification technology platform; the Stem Cell Research Institute is a key laboratory of the hospital and has carried out basic and clinical application research on stem cell and immune cell therapy. The experimental platform meets all the instruments, equipment and technical support required by the present invention, providing a guarantee for the smooth implementation of the project.

[0045] In short, this invention has good feasibility and advancement in terms of clinical design, adopted research plan, and required technical platform. If funded, it can achieve the expected goals.

[0046] The source of exosomes is innovative: From the perspective of breast tumor diagnosis and treatment, this invention innovatively proposes combining the chemotherapy drug cyclophosphamide with breast tumor stem cells to create drug-resistant stem cell lines, upon which exosomes can be isolated. Exosomes, a recent research hotspot in life sciences and translational medicine, offer potential diagnostic markers and therapeutic targets for a variety of diseases. They can also be engineered into highly effective targeted drug delivery vehicles to enable innovative drug development, and hold broad clinical application prospects.

[0047] The present invention combines breast tumor chemotherapy drugs, stem cells and exosomes to prepare drug-loaded breast tumor stem cell exosomes. Technically, proteomics technology, protein expression profiles and database screening of key exosome proteins are preferred, which are different from transcriptome sequencing analysis. This has not been reported in China.

[0048] This invention has transformed the basic research of exosomes into clinical application of new products of key exosome proteins, which will provide experimental basis and reference for further targeted clinical treatment of breast tumor resistance.

[0049] Specific quantitative description (limited to 500 words): Breast cancer is a common and frequently occurring disease that poses a serious threat to human health. The rising morbidity and mortality rates place a heavy burden on Chinese society and healthcare. With the rapid development of modern medicine, the development and screening of novel diagnostic markers with high sensitivity and specificity for breast cancer has become a key area of clinical diagnosis and treatment, addressing the challenges of early diagnosis and prognostic assessment of malignant tumors. Exosomes are attracting significant attention in the biomedical field, particularly in the development and clinical translation of diagnostic technologies related to precision medicine. This study focuses on cancer stem cells and exosomes, applying novel quantitative proteomics techniques to identify key exosomal proteins that are significantly differentially expressed in breast cancer stem cells and engineering them into highly effective targeted drug delivery vehicles. This project demonstrates promising potential as a potential drug delivery vehicle for the treatment of cancer diseases. Furthermore, the detection of exosome markers can dynamically reflect a patient's disease status in real time, offering broad applications in areas such as early disease screening, non-invasive diagnosis, and therapeutic efficacy monitoring.

[0050] The main content of this invention is the application research of drug-loaded exosome biomarkers in the prevention, control, treatment and prognosis of breast tumors, as well as the translational research of developing diagnostic kits. By leveraging the advantages of integrating resources from various disciplines, the research background and content are closely aligned with the guidelines for social development, population and health fields, and the clinical translation and application of key technologies.

[0051] The completion of this project will positively impact the environment and industrialization of chip R&D, and provide the potential for industrialization of chip R&D projects currently under development in the autonomous region. Detection chips are highly functional, cover a wide range of applications, and enjoy significant market demand. Their development is a research hotspot in related fields, offering new insights and effective solutions for laboratory testing and clinical testing.

[0052] Industrial application prospects: Based on the research results of the project, a relatively clear research direction has been condensed. In the future, we will continue to conduct in-depth research on the regulatory relationship between NKD1 and tumor stem cells, clarify the specific molecular regulatory mechanism, and conduct in-depth clinical application research on tumors targeting NKD1, so as to improve the quality and output level of the results and enhance the level of scientific research in the region.

[0053] Through the implementation of the project, we will study new target genes for tumor proliferation and drug resistance, develop related therapeutic drugs, promote the development and improvement of tumor research in Ningxia, and benefit more cancer patients.

[0054] The goal is to discover specific targets for IMPC and establish a diagnosis and treatment system, thereby solving the clinical needs for specific diagnosis and targeted treatment of IMPC. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 This is a flow chart of a method for screening drug-loaded exosome biomarkers derived from breast cancer stem cells provided by an embodiment of the present invention.

[0056] Figure 2 This is a block diagram of the system structure for screening drug-loaded exosome biomarkers derived from breast cancer stem cells provided by an embodiment of the present invention.

[0057] Figure 3 This is a technical roadmap provided by an embodiment of the present invention.

[0058] Figure 4 This is the isolation, culture and identification of breast cancer stem cells provided by the embodiments of the present invention; A: tumor stem cell culture morphology; B: RT-PCR detection of stemness marker expression; C: Western blot detection of stemness marker expression.

[0059] Figure 5 These are the exosome identification results provided by the embodiments of the present invention; A: exosome transmission electron microscopy detection; B: exosome particle size detection.

[0060] Figure 6 : This is the quantitative proteomics detection result provided by the embodiment of the present invention; A: cluster diagram; B: volcano diagram. DETAILED DESCRIPTION

[0061] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0062] like Figure 1 As shown, the method for screening drug-loaded exosome biomarkers derived from breast cancer stem cells provided in an embodiment of the present invention comprises the following steps:

[0063] S101, preparation of drug-loaded breast cancer stem cell exosomes;

[0064] S102, quantitative proteomics screening of key exosome proteins and bioinformatics analysis;

[0065] S103, Study on the biological functions and molecular mechanisms of key exosomal proteins in breast cancer drug resistance;

[0066] S104, Screening and clinical translational application of drug-loaded breast cancer stem cell exosome biomarkers.

[0067] Drug-containing exosomes were obtained by separating the cell culture supernatant from breast cancer stem cells pretreated with chemotherapy drugs (such as docetaxel or paclitaxel) using a combination of ultracentrifugation and density gradient centrifugation. Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) were used to confirm the exosome size distribution (30–150 nm) and bilayer membrane structure. Western blot analysis was performed to detect exosome marker proteins such as CD9, CD63, and TSG101 to verify their purity and integrity.

[0068] Purified drug-loaded exosomes were subjected to TMT (Tandem Mass Tag)-labeled quantitative proteomics analysis, using LC–MS / MS to identify differentially expressed proteins associated with drug response. The resulting differential protein list was entered into protein interaction network databases (such as STRING) and pathway enrichment tools (such as DAVID and KEGG) to screen for candidate exosomal biomarkers significantly associated with functions such as drug resistance, cell survival, and drug efflux, providing targets for subsequent functional validation.

[0069] We selected 1–3 exosomal proteins with the highest enrichment in quantitative analysis and strong associations with multidrug resistance pathways to construct overexpression and siRNA knockdown models. Through in vitro drug sensitivity assays (MTT / CCK-8 cell viability assays), flow cytometry (apoptosis rate and cell cycle analysis), and Western blot analysis (detecting phosphorylation status of P-gp, MDR1, and AKT / ERK pathway proteins), we systematically analyzed the biological functions and signaling transduction mechanisms of these proteins in the development of drug resistance in breast cancer cells.

[0070] Based on these validation results, a multiplex assay kit based on either an ELISA or Luminex platform was developed to quantitatively measure the levels of candidate exosomal proteins in serum samples from 50 breast cancer patients before and after chemotherapy. Receiver operating characteristic (ROC) curves and Kaplan–Meier survival analysis were used to evaluate the diagnostic performance of the kit in predicting chemotherapy resistance and progression-free survival (PFS). Simultaneously, a joint modeling approach was established with imaging and pathological staging data, providing a reliable clinical tool for accurately monitoring the progression of breast cancer drug resistance and tailoring medication.

[0071] Preparation of drug-loaded breast cancer stem cell exosomes provided by the embodiment of the present invention:

[0072] (1) Tissue specimens from clinically diagnosed triple-negative breast cancer patients were collected, primary cells were isolated and cultured through mechanical digestion, and stem cell markers Nanog, OCT4, ALDH1, and ABCG2 were identified to confirm that the cells were derived from breast cancer stem cells.

[0073] (2) Cyclophosphamide, a conventional chemotherapy drug for triple-negative breast cancer patients, was selected and treated with a certain drug concentration in breast cancer stem cells to construct drug-resistant stem cell lines;

[0074] (3) The stem cell culture supernatants from the untreated and treated groups were collected, and exosomes derived from breast cancer stem cells were isolated and identified by density gradient ultracentrifugation;

[0075] (4) Based on the construction of drug-resistant breast cancer stem cell lines, exosomes were isolated and combined to prepare drug-loaded breast cancer stem cell exosomes.

[0076] Quantitative proteomics screening of key exosome proteins and bioinformatics analysis provided by the embodiments of the present invention:

[0077] (1) TMT quantitative proteomics analysis screened differentially expressed proteins in exosomes. Bioinformatics analysis screened functional proteins with significant fold differences and closely related to stem cell regulation. RT-PCR preliminary verification identified key proteins that were closely related to triple-negative breast cancer resistance and were stably differentially expressed.

[0078] (2) Collect blood samples from 100 triple-negative breast cancer patients and normal controls, and use RT-PCR to further verify the universality of the expression of the above key proteins in breast cancer, and preliminarily determine whether they can be used as candidate biomarkers.

[0079] Research on the biological functions and molecular mechanisms of key exosome proteins in breast cancer drug resistance provided by the embodiments of the present invention:

[0080] (1) Construct key protein interference / overexpression lentiviral vectors, transfect breast cancer stem cells / triple-negative breast cancer cell lines, and detect the effects of key proteins on breast cancer stemness regulation by RT-PCR and Western blotting;

[0081] (2) Construct a breast cancer tumor-bearing animal model, inject key exosomal proteins, detect the corresponding phenotypes or the expression of downstream key molecules, and verify the biological functions of key exosomal proteins in breast cancer;

[0082] (3) Bioinformatics prediction of downstream target proteins, and CO-IP / laser confocal microscopy to verify the binding between the protein and the downstream target protein;

[0083] (4) TMT quantitative proteomics KEGG pathway screening of signaling pathways involved in regulation, and the use of pathway blockers / agonists to detect phenotypic changes and explore the molecular mechanism of key proteins in breast tumor resistance.

[0084] Screening and clinical application of drug-loaded breast cancer stem cell exosome biomarkers provided by the embodiments of the present invention:

[0085] (1) Blood samples were collected from 100 patients with triple-negative breast cancer before and after cyclophosphamide chemotherapy. The relative expression of key exosome proteins was detected by RT-PCR and ELISA to verify that key proteins can be used as diagnostic markers for breast cancer resistance.

[0086] (2) Use the TCGA database to search for the expression of key proteins in a large sample of breast cancer tissues and their relationship with patients' overall survival, and evaluate the potential of key proteins as prognostic biomarkers for breast cancer at the clinical level;

[0087] (3) Apply databases and clinical samples combined with basic experiments to verify the functions and mechanisms of key proteins, and contact biopharmaceutical companies to try to develop new products for key proteins, such as key protein diagnostic kits, to assess the patient's disease status through kit testing, in order to achieve clinical translation;

[0088] like Figure 2 As shown, an embodiment of the present invention provides a system for screening drug-loaded exosome biomarkers derived from breast cancer stem cells, comprising:

[0089] Preparation module for the preparation of drug-loaded breast cancer stem cell exosomes;

[0090] Analysis module, used for quantitative proteomics screening of key exosome proteins and bioinformatics analysis;

[0091] Research module for studying the biological functions and molecular mechanisms of key exosome proteins in breast cancer drug resistance;

[0092] Screening module for the screening and clinical translation of drug-loaded breast cancer stem cell exosome biomarkers.

[0093] Another object of the present invention is to provide a computer device comprising a memory and a processor, wherein the memory stores a computer program, and when the computer program is executed by the processor, the processor performs the steps of the method for screening drug-loaded breast cancer stem cell-derived exosome biomarkers.

[0094] Another object of the present invention is to provide a computer-readable storage medium storing a computer program, which, when executed by a processor, causes the processor to perform the steps of the method for screening drug-loaded breast cancer stem cell-derived exosome biomarkers.

[0095] Another object of the present invention is to provide an information data processing terminal, which is used to implement the system for screening exosome biomarkers derived from drug-loaded breast cancer stem cells.

[0096] The present invention is specifically implemented:

[0097] like Figure 3 , Technology Roadmap

[0098] Research methods to be adopted:

[0099] (1) Isolation, culture, and identification of breast tumor stem cells: Patients with triple-negative breast cancer were selected, and breast tumor tissue specimens were obtained after surgery and placed in sterile culture medium. The tissues were transferred to the laboratory, washed with sterile PBS, mechanically minced, and digested with trypsin. After single cells appeared, the tissues were centrifuged, the supernatant was removed, and stem cell culture medium was added to isolate and culture breast tumor stem cells. Flow cytometry was used to detect the expression of CD24 and CD44 surface markers, and RT-PCR and Western blot were used to detect the expression of stem cell markers such as Nanog, OCT4, ALDH1, and ABCG2.

[0100] (2) Exosome isolation and identification: Exosomes were obtained by 100,000 g density gradient ultracentrifugation: the cell supernatant was collected, centrifuged to remove cells, cell debris was removed by centrifugation, exosomes were precipitated, and exosomes were resuspended. Exosomes were identified by exosome transmission electron microscopy (TEM), exosome particle size analysis (NTA), and detection of exosome marker proteins CD9, CD63, Tsg101, and HSP70.

[0101] (3) Study on the biological functions of key proteins in exosomes: construct key protein interference / overexpression lentiviral vectors, transfect breast cancer stem cells, and detect transfection efficiency; RT-PCR and Western blot are used to detect the effects of key proteins on stem cell stemness and the expression of core molecules in signaling pathways; and pathway blockers / agonists are used to detect the recovery effect of key proteins on biological functions.

[0102] (4) Research on the regulatory mechanism of key exosome proteins: predict key proteins binding to each other through String database, verify the binding between proteins and downstream target proteins through CO-IP / laser confocal microscopy; verify the binding mode between the two by epigenetic modification;

[0103] (5) Study on the role of key exosome proteins in mouse tumorigenesis and tumor metastasis: A mouse tumor model was constructed, and the culture supernatant of breast cancer stem cells (negative control) and breast cancer stem cell exosomes were transplanted into the mouse tumor model. The tumor volume, weight and survival curve of the mice were detected. Pathological HE staining was used to observe the morphological changes of each group of mice. IHC staining was used to detect the expression of proliferation marker Ki67, key proteins, and stemness markers ALDH1 and ABCG2. Western analysis was used to detect the expression of core molecules in the signaling pathway. After tail vein injection, small animal in vivo imaging was used to observe the brain metastasis and lung metastasis of the tumor.

[0104] 1. Research basis (accumulated research work and achievements related to the present invention):

[0105] The applicant's main research direction is basic research on tumor stem cells. In the past three years, he has mainly carried out research on the drug resistance of colorectal tumors and breast tumors, and has accumulated rich scientific research experience. As the host, he has successively undertaken the Ningxia Natural Science Foundation, the Autonomous Region Key R&D Plan (Talent Attraction Special Project), and the Ningxia Medical University Clinical College First-Class Discipline Open Project (Key Project). As a main member, he participated in 2 National Natural Science Foundation projects and 1 Autonomous Region Key Project; he has published 5 academic papers, including 4 SCI papers as the first author. The applicant's laboratory has been engaged in biochip and high-throughput sequencing related research for a long time, has obtained a large amount of experimental data, and has rich research experience. In the early stage of the present invention, breast tumor stem cells were isolated and cultured, and stemness was identified by marker detection. Exosomes were isolated on the basis of cultured stem cells, and verified by exosome transmission electron microscopy (TEM) and exosome particle size (NTA) detection. Quantitative proteomics combined with bioinformatics analysis were used to screen for proteins with significant differences in stem cell-derived exosomes. The early results are as follows:

[0106] (1) Isolation, culture and identification of breast cancer stem cells:

[0107] The culture supernatant of the breast tumor cell line MCF-7 was collected and the cells were cultured in vitro for about 10 days to form suspended cell clones (Spheres). After subculture, the cells still maintained the clone sphere shape. RT-PCR and Western blot were used to detect tumor stem cell markers. The results showed that the expression of stem cell markers in Spheres was significantly increased compared with that in adherent MCF-7 cells. Figure 4 .

[0108] (2) Exosome extraction and identification:

[0109] Transmission electron microscopy (TEM) examination revealed that the cells were saucer-shaped, which meets the morphology of exosome cells; the particle size (NTA) was 252nm. The above tests confirmed that the cells were derived from exosomes. Figure 5 .

[0110] (3) Quantitative proteomics screening of key proteins with significant differences in breast cancer stem cell-derived exosomes

[0111] Quantitative proteomics was used to detect key proteins with significant differences in exosomes derived from breast cancer stem cells. Figure 6 A is a cluster diagram, each column represents a sample, and each row represents the expression level of a protein in different samples, red is relatively high expression, and blue is relatively low expression; Figure 6 B is a volcano plot, with the horizontal axis being -log10 (p-values) and the vertical axis being log2 (fold change). Proteins marked in red are upregulated, and proteins marked in blue are downregulated (the volcano plot can intuitively reflect the number and significance of differentially expressed proteins). Figure 6 .

[0112] 2. Working conditions (including existing experimental conditions, lacking experimental conditions and proposed solutions, including plans and implementation of research bases such as national laboratories, state key laboratories, and departmental key laboratories);

[0113] The Biochip Engineering Research Center is equipped with the key experimental conditions and necessary instruments and equipment for this research, including a chip spotting room, chip preparation room, integrated laboratory, sequencing room, verification room, PCR room, and electrophoresis room. The center currently has eight full-time researchers, including three PhDs and four Masters. With clear division of labor and complementary strengths, the center is capable of completing the work of this invention.

[0114] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with this technical field within the technical scope disclosed by the present invention and within the spirit and principles of the present invention should be covered by the scope of protection of the present invention.

Claims

1. A method for screening drug-loaded exosome biomarkers derived from breast cancer stem cells, characterized in that: The following steps are involved: (1) The supernatant of breast cancer stem cells pretreated with chemotherapy drugs was separated by differential centrifugation and density gradient centrifugation to obtain drug-loaded exosomes, and their particle size, morphology, and marker proteins were verified by nanoparticle tracking analysis, transmission electron microscopy, and Western blot. (2) performing TMT-labeled quantitative proteomic analysis on the exosomes, identifying differentially expressed proteins using LC–MS / MS, and screening candidate biomarkers by combining databases such as STRING, DAVID, and KEGG and pathway enrichment tools; (3) Construct candidate protein overexpression and siRNA knockdown cell models, and verify the function and molecular mechanism of candidate proteins in breast cancer resistance through MTT / CCK-8 cell viability assay, flow cytometry apoptosis and cell cycle analysis, and Western blot detection of P-gp / MDR1 and AKT / ERK pathway phosphorylation status; (4) Based on the candidate protein, an ELISA or Luminex multiplex detection kit was constructed to quantitatively detect the candidate protein in the serum of patients before and after breast cancer chemotherapy, and the clinical value of predicting chemotherapy resistance and progression-free survival (PFS) was evaluated by combining ROC curve and Kaplan–Meier survival analysis.

2. The method for screening drug-loaded breast cancer stem cell-derived exosome biomarkers according to claim 1, wherein: Preparation of the drug-loaded breast cancer stem cell exosomes: (1) Tissue specimens from clinically diagnosed triple-negative breast cancer patients were collected, primary cells were isolated and cultured through mechanical digestion, and stem cell markers Nanog, OCT4, ALDH1, and ABCG2 were identified to confirm that the cells were derived from breast cancer stem cells. (2) Cyclophosphamide, a conventional chemotherapy drug for triple-negative breast cancer patients, was selected and treated with a certain drug concentration in breast cancer stem cells to construct drug-resistant stem cell lines; (3) The stem cell culture supernatants from the untreated and treated groups were collected, and exosomes derived from breast cancer stem cells were isolated and identified by density gradient ultracentrifugation; (4) Based on the construction of drug-resistant breast cancer stem cell lines, exosomes were isolated and combined to prepare drug-loaded breast cancer stem cell exosomes.

3. The method for screening drug-loaded breast cancer stem cell-derived exosome biomarkers according to claim 1, wherein: The quantitative proteomics screening of key exosome proteins and bioinformatics analysis: (1) TMT quantitative proteomics analysis screened differentially expressed proteins in exosomes. Bioinformatics analysis screened functional proteins with significant fold differences and closely related to stem cell regulation. RT-PCR preliminary verification identified key proteins that were closely related to triple-negative breast cancer resistance and were stably differentially expressed. (2) Collect blood samples from 100 triple-negative breast cancer patients and normal controls, and use RT-PCR to further verify the universality of the expression of the above key proteins in breast cancer, and preliminarily determine whether they can be used as candidate biomarkers.

4. The method for screening drug-loaded breast cancer stem cell-derived exosome biomarkers according to claim 1, wherein: Study on the biological functions and molecular mechanisms of key exosome proteins in breast cancer drug resistance: (1) Construct key protein interference / overexpression lentiviral vectors, transfect breast cancer stem cells / triple-negative breast cancer cell lines, and detect the effects of key proteins on breast cancer stemness regulation by RT-PCR and Western blotting; (2) Construct a breast cancer tumor-bearing animal model, inject key exosomal proteins, detect the corresponding phenotypes or the expression of downstream key molecules, and verify the biological functions of key exosomal proteins in breast cancer; (3) Bioinformatics prediction of downstream target proteins, and CO-IP / laser confocal microscopy to verify the binding between the protein and the downstream target protein; (4) TMT quantitative proteomics KEGG pathway screening of signaling pathways involved in regulation, and the use of pathway blockers / agonists to detect phenotypic changes and explore the molecular mechanism of key proteins in breast tumor resistance.

5. The method for screening drug-loaded breast cancer stem cell-derived exosome biomarkers according to claim 1, wherein: Screening and clinical transformation application of the drug-loaded breast cancer stem cell exosome biomarkers: (1) Blood samples were collected from 100 patients with triple-negative breast cancer before and after cyclophosphamide chemotherapy. The relative expression of key exosome proteins was detected by RT-PCR and ELISA to verify that the key proteins can be used as diagnostic markers for breast cancer resistance. (2) Use the TCGA database to search for the expression of key proteins in a large sample of breast cancer tissues and their relationship with the overall survival rate of patients, and evaluate the potential of key proteins as prognostic biomarkers for breast cancer at the clinical level; (3) Apply databases and clinical samples combined with basic experiments to verify the functions and mechanisms of key proteins, and contact biopharmaceutical companies to try to develop new key protein products, such as key protein diagnostic kits, to assess the patient's disease status through kit testing, in order to achieve clinical transformation.

6. A system for screening drug-loaded breast cancer stem cell-derived exosome biomarkers for implementing the method for screening drug-loaded breast cancer stem cell-derived exosome biomarkers according to any one of claims 1 to 5, characterized in that: The system for screening drug-loaded breast cancer stem cell-derived exosome biomarkers comprises: Preparation module for the preparation of drug-loaded breast cancer stem cell exosomes; Analysis module, used for quantitative proteomics screening of key exosome proteins and bioinformatics analysis; Research module for studying the biological functions and molecular mechanisms of key exosome proteins in breast cancer drug resistance; Screening module for the screening and clinical translation of drug-loaded breast cancer stem cell exosome biomarkers.

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