Patient-specific bile duct cancer chip and method thereof in dynamic drug resistance evolution analysis

Abstract

The invention discloses a patient-specific bile duct cancer chip and a method thereof in dynamic drug resistance evolution analysis, and relates to the technical field of biomedicine, the patient-specific bile duct cancer chip comprises a central tumor microenvironment chamber, the central tumor microenvironment chamber is internally provided with a plurality of independent three-dimensional culture chambers, and patient-derived cells are embedded and loaded in a matrix; the perfusion channel network is connected with the plurality of three-dimensional culture chambers to form a flow channel and is used for continuously perfusing a culture medium, a medicine or a stromal cell suspension and simulating blood flow and interstitial fluid flow, and the microenvironment regulation and control unit is integrated with the perfusion channel network to control the flow channel. The chip provided by the invention integrates tumor cells, cancer-related fibroblasts and endothelial cells, so that the tumor cells, the cancer-related fibroblasts and the endothelial cells grow in a 3D matrix to form organoids with spatial structures and interstitial infiltration, and the microfluidic perfusion simulates blood / interstitial fluid flow, so that the drug test environment is closer to the actual situation of a human body, and the obtained data has higher physiological prediction value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to patient-specific cholangiocarcinoma chips and their methods in dynamic drug resistance evolution analysis. Background Technology

[0002] Cholangiocarcinoma (CCA) is a highly heterogeneous malignant tumor with a very poor prognosis. Chemotherapy resistance is a major cause of treatment failure, and its occurrence is a dynamic and complex evolutionary process involving the interaction between intrinsic gene alterations in tumor cells and the external microenvironment. Existing microarrays for analyzing patient-specific cholangiocarcinoma drug resistance evolution have the following problems: The simplification and lack of physiological relevance of the chip: Existing 2D cell culture cannot reproduce the 3D structure of tumors, cell-matrix interactions, and key physicochemical gradients (such as oxygen, pH, and drug gradients), leading to distorted drug responses and overestimation of drug efficacy; Existing 3D organoids, although more structurally relevant, usually lack key tumor microenvironment components and are in static culture, which cannot simulate the complex perfusion, material transport, and mechanical forces in vivo. Unable to simulate the dynamics of the treatment process: Existing chip-based drug resistance tests can only reflect the initial sensitivity of tumors and cannot capture how tumor cells develop acquired drug resistance through adaptation, selection and evolution during periodic and intermittent clinical drug administration. Limitations of existing tumor microarrays: Existing microarrays mainly use PDX models and animal models. Among them, although the in vivo environment of patient-derived xenograft models in PDX models is realistic, it takes several months to establish and cannot provide timely guidance for clinical decision-making. In addition, the throughput is low and it is difficult to systematically test multiple treatment strategies. Animal models have species differences. The mouse microenvironment is different from that of humans, and it is difficult to implant human stromal cells. Therefore, there is an urgent need to develop a patient-specific cholangiocarcinoma chip for real-time monitoring and analysis of dynamic drug resistance evolution. Summary of the Invention

[0003] The purpose of this invention is to provide a patient-specific cholangiocarcinoma chip and its method for dynamic drug resistance evolution analysis, so as to solve the problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a patient-specific cholangiocarcinoma chip and its method for dynamic drug resistance evolution analysis, comprising: The central tumor microenvironment chamber contains multiple independent three-dimensional culture chambers for embedding patient-derived cells loaded in a matrix. A perfusion channel network, connected to multiple three-dimensional culture chambers to form flow channels, is used for continuous perfusion of culture medium, drugs or matrix cell suspensions, simulating blood flow and interstitial fluid flow; A microenvironment control unit, which is integrated with the infusion channel network, controls the flow channel.

[0005] Preferably, the central tumor microenvironment chamber is a perfusionable microcolumn array used to support 3D culture; The cell load includes patient-derived bile duct carcinoma organoids and patient-derived cancer-associated fibroblasts, wherein the patient-derived bile duct carcinoma organoids serve as core tumor elements and the patient-derived cancer-associated fibroblasts serve to mimic the intercellular matrix. The matrix is ​​a patient-specific extracellular matrix used to provide biochemical and physical support.

[0006] Preferably, the infusion channel network includes: The central perfusion channel, which runs through the central tumor microenvironment chamber, is used to deliver culture medium, drugs, or immune cells; The lateral channels, located on both sides of the central tumor microenvironment chamber, allow for the pre-culture of patient-derived hepatic sinusoidal endothelial cells, mimicking an incomplete tumor vascular barrier, which allows for the exchange of substances but restricts cell passage. Interstitial fluid channels simulate interstitial fluid flow and drug penetration.

[0007] Preferably, the microenvironment control unit includes: A microfluidic pump, used to precisely control the flow rate and drug delivery timing of different channels; A micro-valve for switching the flow path of different drugs or culture media.

[0008] On the other hand, the present invention also provides a method for using a patient-specific cholangiocarcinoma chip in dynamic drug resistance evolution analysis, which, using any of the above-described patient-specific cholangiocarcinoma chips, includes the following steps: Step 1: Establish a patient-specific cholangiocarcinoma microarray and perform baseline assessment; Step 2: Perform dynamic drug pressure application and adaptive evolution; Step 3: Perform real-time multidimensional monitoring and sampling; Step 4: Analyze the drug resistance mechanism and verify the chip. Step 5: Predict drug resistance pathways and develop treatment strategies.

[0009] Preferably, step 1 specifically includes: Step 1.1: Patient-derived bile duct cancer organs and patient-derived cancer-associated fibroblasts are seeded into the central tumor microenvironment chamber at physiologically relevant ratios, and a stable perfusion culture is established and maintained for 5-7 days to form a stable microenvironment. Step 1.2: Monitor organoid growth and invasion using time-lapse microscopy; assess cell viability using fluorescent dyes; perform RNA-seq, whole-exome sequencing, and proteomics analysis using fixed-point sampling or endpoint analysis on the chip to establish initial gene expression profiles and mutation maps; conduct acute drug exposure tests to test first-line chemotherapy drugs and / or targeted drugs, and calculate IC50 values.

[0010] Preferably, step 2 specifically includes: Step 2.1: In the perfusion culture medium, first-line chemotherapy / targeted drugs are periodically introduced at physiologically relevant concentrations to simulate a clinical treatment cycle; Step 2.2: Based on the chip response, dynamically adjust the drug concentration or introduce second-line drug combinations to simulate clinical adjustment strategies; Step 2.3: Simulate long-term evolution under maintenance therapy or sublethal dose pressure; Step 2.4 involves continuous culturing under drug stress for weeks to months, allowing for natural selection and amplification of drug-resistant clones.

[0011] Preferably, step 3 specifically includes: Step 3.1: Track organoid size, morphology, cell death, and cell migration; integrate pH and oxygen sensing microparticles to monitor microenvironment acidification and hypoxia; periodically collect exfoliated cells or exosomes from the effluent for single-cell RNA-seq or ctDNA analysis to dynamically track clonal evolution. Step 3.2: At a predetermined time point, the contents of the chip are removed, and the changes in cellular heterogeneity and gene expression in different regions are analyzed. The activation status of key signaling pathways is analyzed, and changes in DNA methylation or chromatin accessibility are detected.

[0012] Preferably, step 4 specifically includes: Step 4.1: By comparing sequencing data at different time points, clones with significantly increased frequency under drug stress and their unique gene expression characteristics and mutation profiles were identified. Step 4.2: Upregulated drug efflux pumps, enhanced DNA repair, apoptosis escape, drug target mutation / amplification, bypass signal activation, phenotypic conversion, protective factors secreted by CAFs, matrix sclerosis, and hypoxia-induced stem cell characteristics. Step 4.3: Block the inferred key pathways using small molecule inhibitors or antibodies on the chip, observe whether drug resistance can be reversed, remove the drug-resistant organoids evolved from the chip, perform high-throughput drug screening in traditional culture plates to verify their cross-resistance spectrum, transplant the drug-resistant PDOs evolved from the chip into immunodeficient mice to verify their tumorigenicity and drug resistance in vivo, and conduct correlation analysis with the patient's subsequent clinical progress / relapse.

[0013] Preferably, step 5 specifically includes: Step 5.1: Integrate time-series multi-omics data, construct a computational model, and predict the main drug resistance pathways for specific genotypes / initial microenvironments; Step 5.2: Predict experimental combination therapies on the chip, assess their effectiveness in delaying or preventing the emergence of drug resistance, and directly screen effective second- or third-line treatment options based on the phenotype of the established drug resistance chip.

[0014] The technical effects and advantages of this invention are as follows: The chip provided by this invention integrates tumor cells, cancer-associated fibroblasts, and endothelial cells, enabling them to grow in a 3D matrix to form organoids with spatial structures and interstitial infiltration. Microfluidic perfusion simulates the flow of blood / interstitial fluid, generating physiologically relevant gradients of nutrients, oxygen, and drugs, making the drug testing environment closer to the actual human body and obtaining data with greater physiological predictive value.

[0015] This invention uses programmed microfluidic pumps and valves to precisely simulate clinical drug administration protocols on a chip. This long-term dynamic stimulation, lasting from weeks to months, provides tumor cells with time and selective pressure to adapt and evolve, thereby enabling the in vitro reproduction of the process of acquired drug resistance.

[0016] This invention utilizes patient biopsy tissue to create a functional chip within weeks, which is much faster than the PDX model. It can also be tested in parallel cells of the same patient chip, prospectively predicting the most effective strategies to delay or prevent drug resistance, directly providing decision support for personalized clinical medicine. Furthermore, it can proactively add specific pathway inhibitors to the chip at key points in the drug resistance evolution process to verify their potential to reverse drug resistance. Attached Figure Description

[0017] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 This is a flowchart of the method for dynamic drug resistance evolution analysis using the patient-specific cholangiocarcinoma chip of the present invention. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] This invention provides a patient-specific cholangiocarcinoma chip, comprising a central tumor microenvironment chamber, a perfusion channel network, and a microenvironment regulation unit; specifically: The central tumor microenvironment chamber contains multiple independent three-dimensional culture chambers for embedding patient-derived cells loaded in the matrix. The central tumor microenvironment chamber is a perfusionable microcolumn array used to support 3D culture. The cell load includes patient-derived cholangiocarcinoma organoids and patient-derived cancer-associated fibroblasts. The patient-derived cholangiocarcinoma organoids serve as core tumor elements, while the patient-derived cancer-associated fibroblasts simulate the intercellular matrix. The matrix is ​​a patient-specific extracellular matrix, providing biochemical and physical support. Within the central tumor microenvironment chamber, the microcolumn array allows cells to self-organize into organoids and enables three-dimensional interactions between cells and between cells and the matrix, simulating the in vivo tumor structure and mechanical microenvironment. This overcomes the misjudgment of efficacy caused by cell polarity loss, abnormal contact inhibition, and unrealistic drug diffusion patterns in 2D culture. A perfusion channel network connects to multiple three-dimensional culture chambers to form flow channels for continuous perfusion of culture medium, drugs, or matrix cell suspensions, simulating blood flow and interstitial fluid flow. The perfusion channel network includes: a central perfusion channel that runs through the central tumor microenvironment chamber for delivering culture medium, drugs, or immune cells; lateral channels located on either side of the central tumor microenvironment chamber, capable of pre-culturing patient-derived hepatic sinusoidal endothelial cells to simulate an incomplete tumor vascular barrier, allowing material exchange but restricting cell passage; and interstitial fluid channels to simulate interstitial fluid flow and drug penetration. Within the perfusion channel network, the central channel simulates blood / interstitial fluid flow, continuously delivering nutrients, oxygen, and drugs while removing metabolic waste and maintaining long-term cell viability; the lateral channels simulate incomplete tumor blood vessels, forming a material diffusion barrier and generating physiologically relevant drug, oxygen, and nutrient gradients, thus addressing the problem that static culture, with its homogeneous environment, cannot simulate the gradient microenvironment prevalent in in vivo tumors. The microenvironment control unit is integrated with the perfusion channel network to control the flow channels. The microenvironment control unit includes: a microfluidic pump, which is used to precisely control the flow rate and drug delivery sequence of different channels; and microvalves, which are used to switch between different drug or culture medium flow paths. The microenvironment control unit, composed of the microfluidic pump and microvalves, can programmably realize periodic drug delivery, dose escalation, sequential or combined drug treatment, accurately simulate clinical treatment courses, precisely adjust shear force, and simulate the physiological conditions of different vascular regions.

[0020] The present invention also provides, for example Figure 1 The method for using the patient-specific cholangiocarcinoma chip in dynamic drug resistance evolution analysis, as shown in the above embodiments, includes the following steps: Step 1: Establish a patient-specific cholangiocarcinoma microarray and perform baseline assessment. Use the microarray to create an in vitro three-dimensional model that simulates the tumor microenvironment of human cholangiocarcinoma, serving as the "starting point" or "baseline" for all subsequent experiments. Integrate tumor cells from specific patients into the microarray. These tumor cells can be surgical samples, biopsy specimens, or organoids, preserving the unique genetic, molecular, and phenotypic characteristics of the patient's tumor. Before applying any drug stress, comprehensively assess the initial state of tumor proliferation, apoptosis, gene expression, metabolic activity, and sensitivity to standard drugs, providing a control for subsequent dynamic changes. Step 1 specifically includes: Step 1.1: Patient-derived bile duct cancer organs and patient-derived cancer-associated fibroblasts are seeded into the central tumor microenvironment chamber at physiologically relevant ratios, and a stable perfusion culture is established and maintained for 5-7 days to form a stable microenvironment; thus realizing the transformation from patient tissue to standardized "in vitro microtumor". Step 1.2 involves monitoring organoid growth and invasion, such as CAF-mediated matrix deformation, using time-lapse microscopy; assessing cell viability using fluorescent dyes such as Calcein-AM / PI; performing RNA-seq, whole-exome sequencing, and proteomics analysis via on-chip fixed-point sampling or endpoint analysis to establish initial gene expression profiles and mutation maps; conducting acute drug exposure tests, typically for 48-72 hours, to test first-line chemotherapy drugs and / or targeted drugs, and calculating IC50 values; thereby establishing a multi-dimensional digital profile of the patient's tumor baseline.

[0021] Step 2: Perform dynamic drug pressure application and adaptive evolution; Step 2 specifically includes: Step 2.1: In the perfusion culture medium, first-line chemotherapy / targeted drugs are introduced periodically at physiologically relevant concentrations to simulate the clinical treatment cycle. The specific concentration can be referenced from the patient's plasma Cmax, and the dosing cycle can follow a pattern of 3 days of dosing followed by 4 days of withdrawal. In a controllable and repeatable manner, the chip simulates the real scenario of long-term exposure of tumors to drugs in the patient's body. Through continuous or intermittent sublethal concentration drug pressure, the tumor cell population is actively driven to undergo Darwinian selection or Lamarckian adaptive changes, promoting the emergence and expansion of drug-resistant subpopulations. This establishes a dynamic process from drug sensitivity to the acquisition of drug resistance, providing a time dimension for real-time monitoring. Step 2.2: Based on the chip response, dynamically adjust the drug concentration or introduce second-line drug combinations to simulate clinical adjustment strategies; Step 2.3: Simulate long-term evolution under maintenance therapy or sublethal dose pressure; Step 2.4 involves culturing the drug-resistant clones under pressure for several weeks to months, allowing for natural selection and amplification of the resistant clones; thus, the key selection pressures driving clinical drug resistance can be reproduced in vitro.

[0022] Step 3: Perform real-time multidimensional monitoring and sampling; this allows for the real-time acquisition of multidimensional data during the evolutionary process without disrupting the chip structure and continuity. Simultaneously, it enables the tracking of changes from the molecular, cellular, and tissue levels on the same platform, linking phenotypes and mechanisms at different time points, and performing micro-sampling at key time points for in-depth omics analysis. The time points can be selected from the initial stress phase, the tolerance plateau phase, and the significant drug resistance phase. Step 3 specifically includes: Step 3.1: Track organoid size, morphology, cell death, and cell migration; integrate pH and oxygen sensing microparticles to monitor microenvironment acidification and hypoxia; periodically collect detached cells or exosomes from the effluent for single-cell RNA-seq or ctDNA analysis to dynamically track clonal evolution; this step enables continuous, in-situ, and multi-scale detection of the evolutionary process. Step 3.2: At predetermined time points, such as baseline, week 2, 4, and 8, the contents of the chip are extracted to analyze cellular heterogeneity and gene expression changes in different regions, and to analyze the activation status of key signaling pathways, such as MAPK, PI3K / AKT, and stem cell pathways; changes in DNA methylation or chromatin accessibility are detected, enabling the acquisition of high-resolution molecular panoramic snapshots of key evolutionary nodes.

[0023] Step 4: Perform drug resistance mechanism analysis and chip verification; using the phased samples collected in Step 3, the key molecular pathways, cell subpopulations and microenvironment changes driving drug resistance evolution can be systematically revealed. The drug resistance characteristics found in the chip can be compared with the actual response and biopsy sample analysis results in the patient's subsequent clinical treatment to verify the predictive ability of the model. Step 4 specifically includes: Step 4.1: By comparing sequencing data from different time points, especially scRNA-seq and WES, clones with significantly increased frequency under drug stress and their unique gene expression characteristics and mutation profiles are identified, such as novel FGFR2 kinase domain mutations and TP53 mutation enrichment. This step transforms the correlation data into causal hypotheses. Step 4.2: Upregulated drug efflux pumps, enhanced DNA repair, apoptosis escape, drug target mutation / amplification, bypass signal activation, phenotypic conversion, protective factors secreted by CAFs, matrix sclerosis, and hypoxia-induced stem cell characteristics. Step 4.3 involves using small molecule inhibitors or antibodies, such as in combination with AKT inhibitors, to block the inferred key pathways on the chip and observe whether drug resistance can be reversed. The drug-resistant organoids evolved from the chip are then removed and subjected to high-throughput drug screening in conventional culture plates to verify their cross-resistance profile. The drug-resistant PDOs evolved from the chip are then transplanted into immunodeficient mice (PDX model) to verify their tumorigenicity and drug resistance in vivo, and to perform correlation analysis with the patient's subsequent clinical progression / relapse. In this way, a closed loop from mechanism hypothesis to functional verification can be established, confirming the clinical relevance of the findings.

[0024] Step 5: Predict drug resistance pathways and develop treatment strategies; Based on the evolutionary trajectory and resolved mechanisms observed in the chip, predict other potential drug resistance pathways in the patient's tumor in the future, thereby using the chip as an in vitro surrogate or drug sensitivity testing platform to test subsequent combination therapy regimens against existing and potential drug resistance mechanisms, providing direct experimental evidence for clinicians to develop treatment strategies to overcome drug resistance.

[0025] Step 5 specifically includes: Step 5.1: Integrate time-series multi-omics data and construct a computational model. This model can use existing related technologies such as Bayesian networks and system dynamics models to predict the main drug resistance pathways of specific genotypes in the initial microenvironment. Step 5.2: Predict experimental combination therapies on the chip, evaluate their effectiveness in delaying or preventing the emergence of drug resistance, and directly screen effective second- or third-line treatment options based on the phenotype of the established drug resistance chip; thereby completing the leap from "interpreting the past" to "predicting the future" and achieving a closed loop in clinical translation.

[0026] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A patient-specific cholangiocarcinoma chip, characterized by: Comprising: a central tumor microenvironment chamber, which is internally provided with a plurality of independent three-dimensional culture chambers for embedding patient-derived cells loaded in a matrix; a perfusion channel network connected with the plurality of three-dimensional culture chambers to form a flow channel for continuously perfusing culture medium, drugs or matrix cell suspension, simulating blood flow and interstitial fluid flow; a microenvironment regulation unit integrated with the perfusion channel network to control the flow channel.

2. The patient-specific cholangiocarcinoma chip and its method in dynamic drug resistance evolution analysis according to claim 1, characterized in that: The central tumor microenvironment chamber is a perfusable microcolumn array for supporting 3D culture; The cell load includes patient-derived cholangiocarcinoma organoids and patient-derived cancer-associated fibroblasts, the patient-derived cholangiocarcinoma organoids serving as a core tumor element, and the patient-derived cancer-associated fibroblasts simulating the intercellular stroma; The matrix is a patient-specific extracellular matrix for providing biochemical and physical support.

3. The patient-specific cholangiocarcinoma chip and its method in dynamic drug resistance evolution analysis according to claim 1, wherein: The perfusion channel network comprises: a central perfusion channel passing through the central tumor microenvironment chamber for transporting culture medium, drugs or immune cells; a side channel on both sides of the central tumor microenvironment chamber, capable of pre-culturing patient-derived liver sinus endothelial cells to simulate an incomplete tumor vascular barrier, allowing material exchange but limiting cell passage; an interstitial fluid channel simulating interstitial fluid flow and drug penetration.

4. The patient-specific cholangiocarcinoma chip and its method for dynamic resistance evolution analysis according to claim 1, wherein: The microenvironment regulation unit comprises: a microfluidic pump for precisely controlling the flow rate and drug administration timing of different channels; a micro valve for switching the flow path of different drugs or culture media.

5. A method for the analysis of dynamic resistance evolution in patients using a patient-specific cholangiocarcinoma chip according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: Step 1, establishing a patient-specific cholangiocarcinoma chip and performing baseline evaluation; Step 2, dynamic drug pressure application and adaptive evolution; Step 3, real-time multi-dimensional monitoring and sampling; Step 4, drug resistance mechanism analysis and chip verification; Step 5, predicting drug resistance pathways and developing treatment strategies.

6. The use of the patient-specific cholangiocarcinoma chip according to claim 5 for the analysis of dynamic drug resistance evolution, characterized in that: Step 1 specifically comprises: Step 1.1, inoculating patient-derived cholangiocarcinoma organoids and patient-derived cancer-associated fibroblasts in the central tumor microenvironment chamber at a physiologically relevant ratio, and establishing a stable perfusion culture for 5-7 days to form a stable microenvironment; Step 1.2, monitoring organoid growth and invasion by time-lapse microscopy; evaluating cell viability by fluorescent dye; performing RNA-seq, whole exome sequencing and proteomics analysis by fixed-point sampling or end-point analysis on the chip to establish initial gene expression profiles and mutation maps; performing acute drug exposure testing to test first-line chemotherapy drugs and / or targeted drugs and calculating IC50 values.

7. The method of claim 5, wherein the patient-specific cholangiocarcinoma chip is used in dynamic drug resistance evolution analysis. Step 2 specifically comprises: Step 2.1, periodically introducing first-line chemotherapy / targeted drugs at a physiologically relevant concentration in the perfusion medium to simulate clinical treatment cycles; Step 2.2, dynamically adjusting drug concentration or introducing second-line drug combinations according to chip responses to simulate clinical adjustment strategies; Step 2.3, simulating long-term evolution under maintenance therapy or sub-lethal dose pressure; Step 2.4, continuously culturing for several weeks to several months under drug pressure to allow natural selection and amplification of drug-resistant clones.

8. The method of claim 5, wherein the patient-specific cholangiocarcinoma chip is used in the analysis of dynamic drug resistance evolution. Step 3 specifically comprises: Step 3.1, track organoid size, morphology, cell death, and cell migration; integrate pH and oxygen sensing microparticles to monitor microenvironment acidification and hypoxia; collect shed cells or exosomes from the efflux at regular intervals for single-cell RNA-seq or ctDNA analysis to dynamically track clonal evolution; Step 3.2, at predetermined time points, remove the chip contents to analyze cell heterogeneity and gene expression changes in different regions, analyze the activation status of key signaling pathways, and detect changes in DNA methylation or chromatin accessibility.

9. The method of claim 5, wherein the patient-specific cholangiocarcinoma chip is used in the analysis of dynamic drug resistance evolution. The step 4 specifically includes: Step 4.1, by comparing sequencing data at different time points, identify clones with significantly increased frequency under drug pressure and their unique gene expression characteristics and mutation profiles; Step 4.2, up-regulated drug efflux pumps, DNA repair enhancement, apoptosis escape, drug target mutations / amplification, bypass signal activation, phenotype conversion, protective factors secreted by CAFs, matrix hardening, hypoxia-induced stem cell properties; Step 4.3, use small molecule inhibitors or antibodies on the chip to block the inferred key pathways and observe whether drug resistance can be reversed. Take the evolved drug-resistant organoids out of the chip and perform high-throughput drug screening in traditional culture plates to verify their cross-resistance profiles. Implant the evolved drug-resistant PDOs into immunodeficient mice to verify their tumorigenicity and drug resistance in vivo, and correlate them with the patient's subsequent clinical progression / recurrence.

10. The method of claim 5, wherein the patient-specific cholangiocarcinoma chip is used in the analysis of dynamic drug resistance evolution. The step 5 specifically includes: Step 5.1, integrate time-series multi-omics data to build computational models to predict the main drug resistance pathways under specific genotypes / initial microenvironments; Step 5.2, test experimental combination therapies on the chip to evaluate their effectiveness in delaying or preventing drug resistance. Based on the phenotype of the established drug-resistant chip, directly screen effective second-line or third-line treatment options.

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