Culture system, culture method and application of gastrointestinal tumor chemotherapy drug-resistant organoid

Abstract

The invention discloses a culture system, culture method and application of gastrointestinal tumor chemotherapy drug-resistant organoid, the culture system comprises: a basic culture medium, which is a DMEM / F12 culture medium, and is additionally added with a special growth promoting factor obtained from a natural extract; the multiple growth factors comprise an epidermal growth factor (EGF), a fibroblast growth factor (FGF), a bone morphogenetic protein (BMP) and a vascular endothelial growth factor receptor inhibitor (VEGFRi), and the concentration of each growth factor is accurately screened and optimized; the extracellular matrix is Matrigel modified by physical and chemical methods; the specific additive comprises an antioxidant N-acetylcysteine (NAC). According to the culture system, the culture method and the application of the gastrointestinal tumor chemotherapy drug-resistant organoid, the culture system which is closer to a real environment in vivo is constructed by optimizing basic culture medium components, setting various growth factors and specific additive concentrations and adopting modified Matrigel as an extracellular matrix.

Description

Technical Field

[0001] The present invention relates to the technical field of organoid culture, and in particular to a culture system, culture method and application of chemotherapy-resistant organoids for gastrointestinal tumors. Background Art

[0002] In the field of gastrointestinal tumor treatment, chemotherapy resistance seriously restricts the treatment effect and prognosis of patients. Organoid culture technology provides an important direction for in-depth research on this problem. The invention with the existing published patent number "CN119242586A" has made certain progress in gastrointestinal tumor organoid culture. By adding antioxidant active traditional Chinese medicine extracts, the primary survival rate and proliferation rate of organoids after passage have been improved. However, it still has the following shortcomings in chemotherapy resistance research and clinical application: 1. The culture system of this patent does not adequately simulate the tumor microenvironment and does not consider the interaction between tumor cells and surrounding cells and immune cells, as well as key microenvironmental factors such as tumor hypoxia. In the actual process of tumor development, the tumor microenvironment has a significant impact on the biological behavior of tumor cells and the development of chemotherapy resistance. The "CN119242586A" patent fails to effectively simulate this complex environment, resulting in the cultured organoids being unable to truly reflect the chemotherapy resistance characteristics of clinical tumor cells; 2. Its application scenario is limited to screening small-molecule targeted anti-tumor drugs. However, there are many types of chemotherapy drugs in clinical treatment. In addition to small-molecule targeted drugs, traditional chemotherapy drugs and combination drug regimens also play an important role in the treatment of gastrointestinal tumors. This patent cannot meet the research needs for diversified chemotherapy drugs and combination drug regimens, limiting its application value in clinical tumor treatment research; 3. The "CN119242586A" patent ignores the heterogeneity of individual patient tumors. Tumors from different patients differ in gene expression, protein composition, and metabolic characteristics. These individual differences can lead to different tumor cell sensitivities and resistance mechanisms to chemotherapy drugs. However, the patent does not optimize for individual differences, making it difficult to develop personalized treatment plans and unable to meet the needs of clinical precision treatment. Therefore, we propose a culture system, culture method, and application of chemotherapy-resistant gastrointestinal tumor organoids. Summary of the Invention

[0003] The main purpose of the present invention is to provide a culture system, culture method and application of chemotherapy-resistant gastrointestinal tumor organoids, which can effectively solve the problems in the background technology.

[0004] To achieve the above object, the technical solution adopted by the present invention is: A culture system for gastrointestinal tumor chemotherapy-resistant organoids, comprising: The basic culture medium is DMEM / F12 medium, supplemented with special growth-promoting factors obtained from natural extracts; Multiple growth factors, including epidermal growth factor (EGF), fibroblast growth factor (FGF), bone morphogenetic protein (BMP), and vascular endothelial growth factor receptor inhibitor (VEGFRi), with the concentration of each growth factor precisely screened and optimized; Extracellular matrix, which is Matrigel modified by physical and chemical methods; Specific additives, including the antioxidant N-acetylcysteine ​​(NAC), the small molecule compound Y-27632, and the immunomodulatory factor IL-2.

[0005] Preferably, the concentration of the epidermal growth factor (EGF) is 40-50 ng / mL, the concentration of the fibroblast growth factor (FGF) is 15-20 ng / mL, the concentration of the bone morphogenetic protein (BMP) is 8-10 ng / mL, the concentration of the antioxidant N-acetylcysteine ​​(NAC) is 0.4-0.5 mM, the concentration of the small molecule compound Y-27632 is 8-10 μM, the concentration of the vascular endothelial growth factor receptor inhibitor (VEGFRi) is 8-10 ng / mL, and the concentration of the immunomodulator IL-2 is 3-5 ng / mL.

[0006] By adopting the above technical solution: epidermal growth factor (EGF), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) synergistically activate the key pathways of PI3K / Akt and MAPK at optimized concentrations, regulating cell proliferation and differentiation, and promoting the formation of specific functional structures in organoids; The antioxidant N-acetylcysteine ​​(NAC) scavenges ROS, and the small molecule compound Y-27632 inhibits cell apoptosis, stabilizes cell status, and significantly improves cell survival and organoid formation efficiency; Vascular endothelial growth factor receptor inhibitors (VEGFRi) block VEGF signaling to simulate a hypoxic microenvironment, while the immunomodulatory factor IL-2 activates immune cells to simulate tumor-immune interactions. The two synergistically induce chemotherapy resistance in tumor cells, giving cultured organoids drug resistance properties close to clinical practice. The specific concentration of each component ensures the stability of the biological characteristics of organoids, providing a reliable experimental model and data basis for multi-omics research on chemotherapy resistance mechanisms, chemotherapy drug screening and the formulation of personalized treatment plans.

[0007] Another object of the present invention is to provide a method for culturing gastrointestinal tumor chemotherapy-resistant organoids, comprising the following steps: (1) Tumor tissue acquisition and processing: Fresh gastrointestinal tumor tissue was selected and digested with trypsin, collagenase IV, and hyaluronidase, combined with gentle mechanical shaking and filtration. The temperature, time, and enzyme concentration were dynamically controlled during the digestion process to obtain tumor cells. (2) Cell inoculation and culture: Digested tumor cells are mixed with modified Matrigel in a specific ratio and then inoculated into a culture plate that has been surface-modified with cell adhesion-promoting polypeptide molecules. The plate is then placed in a 37°C constant temperature incubator containing 5% CO2 and saturated humidity. Fresh culture medium containing the components of the culture system is replaced every 2-3 days. Microfluidic technology is used to regulate the nutrients and metabolic waste in the culture system. (3) Induction of chemotherapy resistance: When the organoids grow to a certain size and morphology, low concentrations of chemotherapy drugs are added to the culture medium. Chemotherapy resistance is induced by gradually increasing the drug concentration and combining intermittent drug treatment.

[0008] Preferably, during the tumor tissue processing, trypsin is used for digestion at 37° C. for 10-15 minutes, and then collagenase IV and hyaluronidase are added to continue digestion for 20-30 minutes.

[0009] By using the above technical solution: trypsin is first digested at 37°C for 10-15 minutes to quickly hydrolyze intercellular protein connections, preliminarily separating tumor cells and creating conditions for subsequent digestion. Collagenase IV and hyaluronidase are then used to digest for 20-30 minutes to specifically decompose collagen fibers and hyaluronic acid in the extracellular matrix, further disperse cells, and improve cell acquisition efficiency. Digestion is performed in steps with strict control of time and temperature to avoid excessive damage to cells caused by prolonged action of a single enzyme, retaining cell activity and integrity to the greatest extent possible, ensuring that the obtained tumor cells have good growth and proliferation capabilities, and laying the foundation for the successful cultivation of high-quality chemotherapy-resistant organoids for gastrointestinal tumors.

[0010] Preferably, when the cells are inoculated, the density of the mixture of tumor cells and modified Matrigel is 1×10 5 -1.5×10 5 cells / mL.

[0011] By adopting the above technical solution: this density maintains moderate contact between cells, which can not only promote intercellular signal transmission through paracrine and other means, providing a favorable microenvironment for cell proliferation, but also avoid competition for space and resources caused by overly dense cells, prevent cell growth inhibition, ensure normal cell metabolism and division, and promote the efficient formation of organoids; Modified Matrigel provides cells with a support environment similar to the extracellular matrix in the body. At this density, cells are fully combined with modified Matrigel, which can better play its role in supporting cell attachment, migration and differentiation, maintain normal physiological functions of cells, help organoids grow stably and construct morphology, and ensure the effectiveness and reliability of the culture system.

[0012] Preferably, during the chemotherapy resistance induction process, the chemotherapy drugs include 5-fluorouracil and oxaliplatin, the initial drug concentration is 0.05-0.1 μM, the drug concentration is increased once every 3 days, each increase is 0.05-0.1 μM, and the intermittent drug treatment is 1 day of drug rest after every 3 days of treatment.

[0013] By adopting the above technical solution, 5-fluorouracil and oxaliplatin, both first-line chemotherapy drugs for gastrointestinal cancer, were selected as induction drugs. This ensured that the induced drug-resistant organoids closely matched the chemotherapy resistance of clinical patients, making the research results more clinically valuable. A low initial concentration (0.05-0.1 μM) was gradually increased (by 0.05-0.1 μM every 3 days) to simulate the gradual accumulation of drug concentration in tumor cells during chemotherapy in vivo, prompting tumor cells to gradually adapt to drug pressure and induce drug resistance mechanisms. Intermittent treatment (with 1 day of drug rest every 3 days of treatment) simulated the intervals between clinical chemotherapy. During the alternating drug treatment and recovery period, tumor cells were screened for cell populations with stronger drug resistance, thus forming a stable chemotherapy-resistant organoid model. This induction method avoids irreversible damage to cells caused by a sudden increase in drug concentration, maintains cell activity and proliferation ability, and ensures that the induced chemotherapy-resistant organoids have stable drug resistance characteristics, providing a reliable experimental model basis for subsequent chemotherapy resistance mechanism research, drug screening and personalized treatment plan formulation.

[0014] Another object of the present invention is to provide a culture system and method for culturing gastrointestinal tumor chemotherapy-resistant organoids, including: Research on the mechanism of chemotherapy resistance; Chemotherapy drug screening; Personalized treatment plan development.

[0015] Preferably, in the study of chemotherapy resistance mechanisms, key genes, signaling pathways, proteins, and metabolites related to chemotherapy resistance are screened by performing transcriptomics, proteomics, and metabolomics multi-omics combined analysis on cultured chemotherapy-resistant organoids and non-resistant organoids.

[0016] By adopting the above technical solutions: transcriptomics can obtain global information on gene expression, proteomics can analyze changes related to drug resistance at the protein level, and metabolomics can present the dynamic changes of metabolites. Multi-omics combined analysis breaks through the limitations of single technologies and comprehensively reveals the molecular mechanisms of chemotherapy resistance in tumor cells from multiple levels of gene transcription, protein synthesis and modification, and metabolic network remodeling, avoiding information omissions. By integrating multi-omics data, we can screen out key genes, signaling pathways, proteins, and metabolites closely related to chemotherapy resistance, locate the core targets for the occurrence and development of resistance, provide clear clues for a deeper understanding of the resistance mechanism, and point the way for the development of targeted strategies to reverse resistance. Multi-omics data corroborate and complement each other, verifying research results from different angles, effectively reducing errors and false positives that are prone to occur with a single technology, significantly improving the reliability and credibility of research results, and laying a solid foundation for subsequent research on drug resistance mechanisms and clinical translation applications.

[0017] Preferably, in chemotherapy drug screening, different chemotherapy drugs are applied to cultured chemotherapy-resistant organoids, and the drug efficacy is comprehensively evaluated by detecting multiple indicators such as the growth inhibition rate, cell apoptosis rate, and metabolic activity changes of the organoids.

[0018] By adopting this technical solution, using cultured chemotherapy-resistant organoids as research subjects, we can highly replicate the chemotherapy-resistant state of tumor cells in vivo. Compared with traditional cell line models, this can more realistically reflect the effects of drugs on drug-resistant tumor cells in the complex environment of the human body, making drug screening results closer to clinical practice. By testing multiple indicators such as organoid growth inhibition rate, cell apoptosis rate, and metabolic activity changes, we can comprehensively and deeply evaluate drug efficacy from different dimensions of tumor cell proliferation, death, and metabolism. Multiple indicators complement each other and verify each other, avoiding the limitations of a single indicator. This allows for more accurate judgment of drug effects on chemotherapy-resistant organoids and screening for truly effective chemotherapy drugs. The multi-index comprehensive evaluation based on chemotherapy-resistant organoids can quickly eliminate ineffective drugs, focus on potentially effective drugs, and greatly improve the efficiency of drug screening. At the same time, the comprehensive analysis of multiple indicators effectively reduces the error and uncertainty of the screening results, enhances the reliability of the screening results, and provides a scientific and accurate basis for the selection of clinical chemotherapy drugs.

[0019] Preferably, in the development of personalized treatment plans, individualized chemotherapy-resistant organoids are cultured using the culture system and method for gastrointestinal tumor tissues from different patients, and then the individualized chemotherapy-resistant organoids are subjected to multi-omics analysis, including but not limited to genomic, transcriptomic, proteomic, and metabolomic analysis, to obtain the molecular characteristics of the patient's tumor cells; Based on the acquired molecular features and combined with the treatment effect data of patients with similar molecular features in the clinical database, we can screen out chemotherapy drugs and combination therapy regimens that may be effective for the patient; By applying the screened drugs and combination drug regimens to individualized chemotherapy-resistant organoids to simulate the in vivo treatment process, the organoids' growth inhibition rate, cell apoptosis rate, resistance-related protein expression and other indicators are re-tested to evaluate the effectiveness of the drugs and regimens, and ultimately develop a personalized gastrointestinal tumor chemotherapy treatment plan for the patient.

[0020] By employing this technology, personalized chemotherapy-resistant organoids are cultivated from tumor tissues derived from different patients, fully accounting for differences in genes, proteins, and metabolism between patients' tumors. This allows for the acquisition of unique molecular signatures, breaking through the limitations of traditional treatments that often require only one treatment for each patient, and enabling treatment plans to be more tailored to the patient's specific condition. Combined with the treatment effect data of patients with similar molecular characteristics in the clinical database, effective chemotherapy drugs and combination therapy regimens are screened. Combining clinical experience with individual patient characteristics provides a scientific basis for the selection of treatment regimens, thereby improving the rationality and effectiveness of treatment regimens. By applying the screening scheme to personalized organoids to simulate the in vivo treatment process, the efficacy is evaluated from multiple indicators such as growth inhibition rate, cell apoptosis rate, and expression of drug-resistance-related proteins, which can intuitively reflect the actual effects of drugs and schemes on the patient's tumor cells, avoid ineffective treatment, ensure that the final personalized treatment plan is feasible, and improve the accuracy of gastrointestinal tumor treatment and patient prognosis.

[0021] Compared with the prior art, the present invention has the following beneficial effects: 1. In this invention, by optimizing the basal culture medium components, setting the concentrations of multiple growth factors and specific additives, and using modified Matrigel as the extracellular matrix, a culture system that is closer to the real in vivo environment is constructed. The vascular endothelial growth factor receptor inhibitor (VEGFRi) blocks VEGF signaling, simulating the hypoxic tumor microenvironment, and the immunomodulatory factor IL-2 activates immune cells, simulating the interaction between the tumor and the immune system, and synergistically inducing chemotherapy resistance in tumor cells. This can more realistically reproduce the chemotherapy resistance process of tumor cells in vivo, providing a more reliable model for the study of chemotherapy resistance mechanisms. 2. The cultured gastrointestinal tumor chemotherapy-resistant organoids in this invention can be widely used in the study of chemotherapy resistance mechanisms, chemotherapy drug screening, and the development of personalized treatment plans. In terms of chemotherapy drug screening, they can not only evaluate traditional chemotherapy drugs such as 5-fluorouracil and oxaliplatin, but also systematically evaluate multiple chemotherapy drugs and combination drug regimens. In the study of chemotherapy resistance mechanisms, through the combined analysis of multi-omics such as transcriptomics, proteomics, and metabolomics, the molecular mechanisms of chemotherapy resistance can be systematically revealed from multiple levels of genes, proteins, and metabolism, greatly expanding the application scenarios and providing more comprehensive technical support for clinical tumor treatment research. 3. In the present invention, individualized chemotherapy-resistant organoids are cultured for gastrointestinal tumor tissues derived from different patients, and the molecular characteristics of tumor cells are obtained through multi-omics analysis. Based on these molecular characteristics, combined with the treatment effect data of patients with similar molecular characteristics in the clinical database, chemotherapy drugs and combination drug regimens that may be effective for the patient are screened out. Then, the screened regimens are applied to the individualized chemotherapy-resistant organoids to simulate the in vivo treatment process, and the effectiveness of the drugs and regimens is further evaluated. Finally, a personalized gastrointestinal tumor chemotherapy treatment plan is formulated for the patient, which fully takes into account the heterogeneity of individual tumors of patients, significantly improves the targetedness and effectiveness of treatment, and promotes the development of gastrointestinal tumor treatment towards precision. DETAILED DESCRIPTION

[0022] The present invention is further described below with reference to the embodiments, but the scope of protection claimed in the present invention is not limited to the scope described in the embodiments.

[0023] Example 1 A culture system for gastrointestinal tumor chemotherapy-resistant organoids, comprising: The basic culture medium is DMEM / F12 medium, supplemented with special growth-promoting factors obtained from natural extracts; Multiple growth factors, including epidermal growth factor (EGF), fibroblast growth factor (FGF), bone morphogenetic protein (BMP), and vascular endothelial growth factor receptor inhibitor (VEGFRi), with the concentration of each growth factor precisely screened and optimized; Extracellular matrix, which is Matrigel modified by physical and chemical methods; Specific additives, including the antioxidant N-acetylcysteine ​​(NAC), the small molecule compound Y-27632, and the immunomodulatory factor IL-2.

[0024] In this embodiment, the concentration of epidermal growth factor (EGF) is 40-50 ng / mL, the concentration of fibroblast growth factor (FGF) is 15-20 ng / mL, the concentration of bone morphogenetic protein (BMP) is 8-10 ng / mL, the concentration of antioxidant N-acetylcysteine ​​(NAC) is 0.4-0.5 mM, the concentration of small molecule compound Y-27632 is 8-10 μM, the concentration of vascular endothelial growth factor receptor inhibitor (VEGFRi) is 8-10 ng / mL, and the concentration of immunomodulatory factor IL-2 is 3-5 ng / mL.

[0025] Through the above scheme: the concentration of epidermal growth factor (EGF) is 40-50ng / mL, which binds to EGFR to activate PI3K / Akt and MAPK pathways, promoting cell proliferation. This concentration ensures sufficient signal activation and avoids abnormal cell proliferation. Fibroblast growth factor (FGF) at a concentration of 15-20 ng / mL synergizes with epidermal growth factor (EGF) to activate the PLCγ / PKC and MAPK pathways, enhancing cell proliferation and inducing tumor cell differentiation into organoids; Bone morphogenetic protein (BMP) at a concentration of 8-10 ng / mL binds to receptors to activate the Smad pathway, regulating cell differentiation and promoting the formation of specific functional structures in organoids. The concentration of the antioxidant N-acetylcysteine ​​(NAC) is 0.4-0.5mM, and its sulfhydryl group scavenges ROS, reduces oxidative stress, protects biomacromolecules, and improves cell survival.

[0026] The small molecule compound Y-27632, at a concentration of 8-10 μM, inhibits ROCK activity, reduces cell apoptosis, stabilizes the cytoskeleton, and improves organoid formation efficiency; The concentration of vascular endothelial growth factor receptor inhibitor (VEGFRi) is 8-10 ng / mL, which blocks VEGF signaling, simulates the tumor hypoxic microenvironment, and induces chemotherapy resistance in cells; The concentration of the immunomodulatory factor IL-2 is 3-5 ng / mL, which activates immune cells, simulates the interaction between tumors and the immune system, and prompts tumor cells to develop chemotherapy resistance mechanisms.

[0027] Example 2 Based on Example 1, a method for culturing chemotherapy-resistant gastrointestinal tumor organoids comprises the following steps: (1) Tumor tissue acquisition and processing: Fresh gastrointestinal tumor tissue was selected and digested with trypsin, collagenase IV, and hyaluronidase, combined with gentle mechanical shaking and filtration. The temperature, time, and enzyme concentration were dynamically controlled during the digestion process to obtain tumor cells. (2) Cell inoculation and culture: Digested tumor cells are mixed with modified Matrigel in a specific ratio and then inoculated into a culture plate that has been surface-modified with cell adhesion-promoting polypeptide molecules. The plate is then placed in a 37°C constant temperature incubator containing 5% CO2 and saturated humidity. Fresh culture medium containing culture system components is replaced every 2-3 days. Microfluidic technology is used to regulate nutrients and metabolic waste in the culture system. (3) Induction of chemotherapy resistance: When the organoids grow to a certain size and morphology, low concentrations of chemotherapy drugs are added to the culture medium. Chemotherapy resistance is induced by gradually increasing the drug concentration and combining intermittent drug treatment.

[0028] In this embodiment, during the tumor tissue processing, trypsin was digested at 37°C for 10-15 minutes, and then collagenase IV and hyaluronidase were added to continue digestion for 20-30 minutes; when cells were seeded, the density of tumor cells mixed with modified Matrigel was 1×10 5 -1.5×10 5 cells / mL; during the induction of chemotherapy resistance, chemotherapy drugs included 5-fluorouracil and oxaliplatin, with an initial drug concentration of 0.05-0.1 μM. The drug concentration was increased every 3 days, with each increase of 0.05-0.1 μM. Intermittent drug treatment was 1 day of drug rest after every 3 days of treatment.

[0029] According to the above protocol: During tumor tissue processing, trypsin is digested at 37°C for 10-15 minutes. This enzyme can specifically hydrolyze protein connections between cells and quickly separate cells. However, prolonged digestion can cause excessive cell damage. Collagenase IV and hyaluronidase are then added to continue digestion for 20-30 minutes. Collagenase IV can break down collagen fibers in the extracellular matrix, and hyaluronidase can degrade hyaluronic acid. The two work synergistically to fully digest the complex extracellular matrix components in tumor tissue, thereby gently and efficiently obtaining tumor cells, ensuring cell activity and integrity, and laying the foundation for subsequent organoid culture. When cells were seeded, the density of tumor cells mixed with modified Matrigel was 1×10 5 -1.5×10 5 cells / mL. This density ensures moderate cell-to-cell interaction, promotes cell proliferation and organoid formation, while avoiding problems such as nutrient competition and metabolic waste accumulation caused by overly dense cells, which affect cell growth. At the same time, modified Matrigel simulates the extracellular matrix environment in the body, providing a scaffold for cell attachment and growth. The appropriate cell density combined with modified Matrigel helps maintain the normal physiological function of cells and the stable development of organoids. During the induction of chemotherapy resistance, 5-fluorouracil and oxaliplatin were selected as chemotherapy drugs. Both are commonly used chemotherapy drugs in the clinical treatment of gastrointestinal tumors. Using them to induce resistance is closer to the actual clinical situation. The initial drug concentration was set at 0.05-0.1μM. The lower concentration of drugs first exerted slight pressure on the tumor cells, allowing the cells to gradually adapt to the drug environment. The drug concentration was increased once every 3 days, each time increasing by 0.05-0.1μM, and the drug pressure was gradually increased to simulate the process of gradual accumulation of drug concentration in tumor cells during chemotherapy in vivo. Intermittent drug treatment was 1 day of drug suspension for every 3 days of treatment. This method can cause tumor cells to produce a stress response during drug treatment, try to adapt to drug pressure and form a resistance mechanism. During the drug suspension period, the cells restored some physiological functions, and cells with stronger drug resistance were screened out, thereby effectively inducing stable chemotherapy-resistant organoids.

[0030] Example 3 Based on Example 1 and Example 2, a culture system and a culture method for gastrointestinal tumor chemotherapy-resistant organoids are used, including: Research on the mechanism of chemotherapy resistance; Chemotherapy drug screening; Personalized treatment plan development.

[0031] In this embodiment, in the study of chemotherapy resistance mechanism, transcriptomics, proteomics, metabolomics and other multi-omics joint analysis is performed on cultured chemotherapy-resistant organoids and non-resistant organoids to screen key genes, signaling pathways, proteins and metabolites related to chemotherapy resistance; in chemotherapy drug screening, different chemotherapy drugs are applied to cultured chemotherapy-resistant organoids, and the drug efficacy is comprehensively evaluated by detecting multiple indicators such as growth inhibition rate, cell apoptosis rate, and metabolic activity change of the organoids; in the formulation of personalized treatment plans, personalized chemotherapy-resistant organoids are cultured using culture systems and methods for gastrointestinal tumor tissues from different patients, and then personalized chemotherapy is performed. Multi-omics analysis, including but not limited to genomic, transcriptomic, proteomic and metabolomic analysis, is performed on drug-resistant organoids to obtain the molecular characteristics of the patient's tumor cells. Based on the obtained molecular characteristics and combined with the treatment effect data of patients with similar molecular characteristics in the clinical database, chemotherapy drugs and combination drug regimens that may be effective for the patient are screened out. By applying the screened drugs and combination drug regimens to individualized chemotherapy-resistant organoids to simulate the in vivo treatment process, the organoids' growth inhibition rate, cell apoptosis rate, resistance-related protein expression and other indicators are re-tested to evaluate the effectiveness of the drugs and regimens, and ultimately develop a personalized gastrointestinal tumor chemotherapy treatment plan for the patient.

[0032] Through the above approach: In the study of chemotherapy resistance mechanisms, transcriptomics can reflect the dynamic changes in gene expression levels, proteomics can analyze protein expression, modification and interactions, and metabolomics can present changes in the types and content of metabolites. By conducting multi-omics joint analysis of chemotherapy-resistant organoids and non-resistant organoids, it is possible to systematically reveal the molecular mechanisms of chemotherapy resistance in tumor cells from multiple dimensions such as gene transcription, protein synthesis and regulation, and metabolic networks. It can also screen key genes related to chemotherapy resistance, clarify the signaling pathways in which they participate, and target specific proteins and metabolites, providing comprehensive information for a deeper understanding of resistance mechanisms and laying the foundation for the development of strategies to reverse resistance. In chemotherapy drug screening, different chemotherapy drugs are applied to chemotherapy-resistant organoids. The drug efficacy is comprehensively evaluated by detecting multiple indicators such as growth inhibition rate, cell apoptosis rate, and metabolic activity changes in the organoids. This has important biological significance. The growth inhibition rate directly reflects the degree of drug inhibition on tumor cell proliferation, the cell apoptosis rate reflects the drug's ability to induce tumor cell death, and the metabolic activity changes can reflect the state of intracellular material metabolism and energy metabolism, which are closely related to cell survival and proliferation. These indicators evaluate the effect of drugs on drug-resistant tumor cells from different levels. Through comprehensive analysis of multiple indicators, drugs with significant inhibitory effects on chemotherapy-resistant organoids can be more accurately screened, providing a reliable basis for the selection of clinical chemotherapy drugs. In the formulation of personalized treatment plans, personalized chemotherapy-resistant organoids are cultured for gastrointestinal tumor tissues from different patients, and multi-omics analysis is combined to obtain the molecular characteristics of the patient's tumor cells. This is the key basis for personalized treatment. Since tumors of different patients differ at the genetic, protein and metabolic levels, based on molecular characteristics combined with the treatment effect data of patients with similar molecular characteristics in the clinical database, chemotherapy drugs and combination drug regimens that may be effective for the patient can be screened out. Then, by applying the screened regimens to personalized chemotherapy-resistant organoids, simulating the in vivo treatment process, and detecting indicators such as the growth inhibition rate, cell apoptosis rate, and resistance-related protein expression of the organoids, the effectiveness of the drugs and regimens can be further evaluated. This treatment plan formulation method based on the individual tumor characteristics of patients can fully consider individual differences, improve the targetedness and effectiveness of treatment, and achieve precise and personalized chemotherapy treatment for gastrointestinal tumors.

[0033] In summary, the present invention optimizes the basal culture medium components, sets the concentrations of multiple growth factors and specific additives, and combines them with a modified extracellular matrix to synergistically promote tumor cell proliferation and differentiation, simulate a chemotherapy-resistant microenvironment, and ensure the stable growth of organoids and the induction of drug-resistant characteristics. In terms of culture methods, a step-by-step optimized process of tissue processing, cell seeding and culture, and chemotherapy resistance induction is adopted to balance cell activity and organoid culture efficiency to ensure the successful construction of drug-resistant organoids; At the application level, we will conduct in-depth research on the mechanism of chemotherapy resistance through multi-omics combined analysis, screen effective chemotherapy drugs through comprehensive multi-index evaluation, and formulate personalized treatment plans based on the molecular characteristics of individual tumors, thus achieving full coverage from mechanism research to clinical application. The overall technical solution provides an efficient model for the study of chemotherapy resistance in gastrointestinal tumors and a scientific basis for clinical treatment, which is of great significance for improving the level of gastrointestinal tumor treatment.

[0034] The above description is merely a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiment. All technical solutions based on the concept of the present invention are within the scope of protection of the present invention. It should be noted that for those skilled in the art, various improvements and modifications that do not depart from the principles of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A culture system for gastrointestinal tumor chemotherapy-resistant organoids, characterized in that: include: The basic culture medium is DMEM / F12 medium, supplemented with special growth-promoting factors obtained from natural extracts; Multiple growth factors, including epidermal growth factor (EGF), fibroblast growth factor (FGF), bone morphogenetic protein (BMP), and vascular endothelial growth factor receptor inhibitor (VEGFRi), with the concentration of each growth factor precisely screened and optimized; Extracellular matrix, which is Matrigel modified by physical and chemical methods; Specific additives, including the antioxidant N-acetylcysteine ​​(NAC), the small molecule compound Y-27632, and the immunomodulatory factor IL-2.

2. The culture system for gastrointestinal tumor chemotherapy-resistant organoids according to claim 1, characterized in that: The concentration of the epidermal growth factor (EGF) is 40-50 ng / mL, the concentration of the fibroblast growth factor (FGF) is 15-20 ng / mL, the concentration of the bone morphogenetic protein (BMP) is 8-10 ng / mL, the concentration of the antioxidant N-acetylcysteine ​​(NAC) is 0.4-0.5 mM, the concentration of the small molecule compound Y-27632 is 8-10 μM, the concentration of the vascular endothelial growth factor receptor inhibitor (VEGFRi) is 8-10 ng / mL, and the concentration of the immunomodulator IL-2 is 3-5 ng / mL.

3. The method for culturing chemotherapy-resistant gastrointestinal tumor organoids according to claim 1, characterized in that: The following steps are included: (1) Tumor tissue acquisition and processing: Fresh gastrointestinal tumor tissue was selected and digested with trypsin, collagenase IV, and hyaluronidase, combined with gentle mechanical shaking and filtration. The temperature, time, and enzyme concentration were dynamically controlled during the digestion process to obtain tumor cells. (2) Cell inoculation and culture: Digested tumor cells are mixed with modified Matrigel in a specific ratio and then inoculated into a culture plate that has been surface-modified with cell adhesion-promoting polypeptide molecules. The plate is then placed in a 37°C constant temperature incubator containing 5% CO2 and saturated humidity. Fresh culture medium containing the components of the culture system is replaced every 2-3 days. Microfluidic technology is used to regulate the nutrients and metabolic waste in the culture system. (3) Induction of chemotherapy resistance: When the organoids grow to a certain size and morphology, low concentrations of chemotherapy drugs are added to the culture medium. Chemotherapy resistance is induced by gradually increasing the drug concentration and combining intermittent drug treatment.

4. The method for culturing chemotherapy-resistant gastrointestinal tumor organoids according to claim 3, characterized in that: During the tumor tissue processing, trypsin is used for digestion at 37° C. for 10-15 minutes, and then collagenase IV and hyaluronidase are added to continue digestion for 20-30 minutes.

5. The method for culturing chemotherapy-resistant gastrointestinal tumor organoids according to claim 3, characterized in that: When the cells were inoculated, the density of the mixture of tumor cells and modified Matrigel was 1×10 5 -1.5×10 5 cells / mL.

6. The method for culturing chemotherapy-resistant gastrointestinal tumor organoids according to claim 3, characterized in that: During the chemotherapy resistance induction process, the chemotherapy drugs include 5-fluorouracil and oxaliplatin, the initial drug concentration is 0.05-0.1 μM, the drug concentration is increased once every 3 days, each increase is 0.05-0.1 μM, and the intermittent drug treatment is 1 day of drug rest after every 3 days of treatment.

7. Use of a culture system and culture method for gastrointestinal tumor chemotherapy-resistant organoids according to claims 1 and 3, characterized in that: include: Research on the mechanism of chemotherapy resistance; Chemotherapy drug screening; Personalized treatment plan development.

8. Use of a method for culturing chemotherapy-resistant gastrointestinal tumor organoids according to claim 7, characterized in that: In the study of chemotherapy resistance mechanisms, we screened key genes, signaling pathways, proteins, and metabolites related to chemotherapy resistance by conducting multi-omics combined analysis of transcriptomics, proteomics, and metabolomics on cultured chemotherapy-resistant organoids and non-resistant organoids.

9. The use of the method for culturing chemotherapy-resistant gastrointestinal tumor organoids according to claim 7, characterized in that: In chemotherapy drug screening, different chemotherapy drugs are applied to cultured chemotherapy-resistant organoids, and the drug efficacy is comprehensively evaluated by detecting multiple indicators such as the growth inhibition rate, cell apoptosis rate, and metabolic activity changes of the organoids.

10. The use of the method for culturing chemotherapy-resistant gastrointestinal tumor organoids according to claim 7, characterized in that: In the development of personalized treatment plans, the culture system and method are used to culture personalized chemotherapy-resistant organoids from gastrointestinal tumor tissues derived from different patients. The personalized chemotherapy-resistant organoids are then subjected to multi-omics analysis, including but not limited to genomic, transcriptomic, proteomic, and metabolomic analysis, to obtain the molecular characteristics of the patient's tumor cells. Based on the acquired molecular features and combined with the treatment effect data of patients with similar molecular features in the clinical database, we can screen out chemotherapy drugs and combination therapy regimens that may be effective for the patient; By applying the screened drugs and combination drug regimens to individualized chemotherapy-resistant organoids to simulate the in vivo treatment process, the organoids' growth inhibition rate, cell apoptosis rate, resistance-related protein expression and other indicators are re-tested to evaluate the effectiveness of the drugs and regimens, and ultimately develop a personalized gastrointestinal tumor chemotherapy treatment plan for the patient.