Method for screening anti-angiogenesis combined medicine by using vascularization co-culture organ chip

Abstract

The invention relates to the technical field of drug screening, in particular to a method for screening an anti-angiogenesis combined drug by using a vascularization co-culture organ chip. The method comprises the following specific steps: S1, screening candidate drugs with potential sensitivity to indications based on an open source database, and carrying out cell in-vitro screening; s2, constructing a vascularized co-culture organ chip; s3, using the vascularized co-culture organ chip constructed in the step S2 to perform drug sensitivity detection of an anti-tumor drug combination scheme; and S4, screening out tumor growth inhibiting drugs in the drug combination according to a drug sensitivity detection result, and taking the drugs as alternative drugs with potential combination effects. Drug sensitivity screening is carried out based on open source data and the vascularized organ chip simulating the tumor microenvironment, the screening accuracy of an angiogenesis inhibiting drug combination scheme is improved, and the optimal angiogenesis inhibiting drug combination scheme is screened out.

Description

Technical Field

[0001] The present invention relates to the technical field of drug screening, and in particular to a method for screening anti-angiogenesis combination drugs by utilizing vascularized co-cultured organ chips. Background Art

[0002] The use of anti-angiogenic drugs in tumor treatment has a history of decades. By inhibiting the formation of new blood vessels, these drugs can cut off the blood supply to the tumor, limiting its growth and spread. At present, the commonly used anti-angiogenic drugs in clinical practice mainly include bevacizumab, ranibizumab and apatinib. In monotherapy, anti-angiogenic drugs have shown certain efficacy. For example, bevacizumab has been widely used in the treatment of metastatic colorectal cancer and non-small cell lung cancer. However, monotherapy also has some problems, including drug resistance and side effects. These problems limit the further improvement of its clinical efficacy.

[0003] In order to overcome the limitations of monotherapy, researchers have tried to combine angiogenesis-inhibiting drugs with other types of anti-tumor drugs. Common combination therapy regimens include combinations of angiogenesis-inhibiting drugs with chemotherapy drugs, immune checkpoint inhibitors, targeted therapy drugs, etc. These combination therapy regimens have shown better efficacy in some patients, but most of them are based on experience and clinical trials, and are also accompanied by more complex risks of drug resistance and treatment responses. Existing preclinical drug research tools are mainly based on two-dimensional cell culture and animal models, among which two-dimensional cells cannot simulate the tumor microenvironment and animal models cannot achieve high-throughput detection, lacking systematic and efficient screening methods. In order to improve screening efficiency and accuracy, it is particularly important to develop a high-throughput screening platform based on in vitro models.

[0004] Organ chip technology has been a hot topic in tumor research in recent years. Organ chip is a three-dimensional culture system that can simulate the microenvironment and tissue structure of tumors. In organ chips, tumor cells can more realistically display their biological characteristics. Vascularized co-culture technology achieves the formation of vascular networks in organoids by co-culturing endothelial cells and tumor organoids in the same organ chip system. This technology provides a more realistic model for simulating the tumor microenvironment and can better evaluate the efficacy and safety of drugs. Summary of the invention

[0005] The purpose of the present invention is to address the problems existing in the background technology and propose a method for screening anti-angiogenic combination drugs using vascularized co-culture organ chips, to perform drug sensitivity screening based on open source data and vascularized organ chips that simulate the tumor microenvironment, to improve the accuracy of screening anti-angiogenic drug combination schemes, and to screen out the best anti-angiogenic drug combination regimen.

[0006] The technical solution of the present invention is a method for screening anti-angiogenic combination drugs using vascularized co-cultured organ chips, comprising the following specific steps:

[0007] S1. Screen candidate drugs with potential sensitivity to indications based on open source databases and perform in vitro cell screening;

[0008] Indication types include lung cancer, colorectal cancer, liver cancer, pancreatic cancer or gastric cancer; collect drugs from open source databases that have completed indication-related cell lines, including IC50 values ​​of sensitivity test data for inhibiting angiogenesis and inhibiting tumor growth, and Cmax values ​​of blood drug concentration data of corresponding drugs, and compare the two; the drug sensitivity test data comes from the GDSC database (https: / / www.cancerrxgene.org / ), and the maximum blood drug concentration Cmax value of the drug comes from the DrugBank database (https: / / go.drugbank.com / ); perform in vitro cell line screening on candidate drugs screened out from the database, obtain IC50 values ​​of sensitivity test data for inhibiting angiogenesis and inhibiting tumor growth, and compare them with Cmax values ​​of blood drug concentration data of corresponding drugs, verify the accuracy of database screening, further screen candidate drugs, and eliminate insensitive drugs.

[0009] S2. Construction of vascularized co-culture organ chips;

[0010] S21, Organoid preparation and seeding;

[0011] S211. Sample collection: Collect multiple different human tumor tissue samples to maintain tissue activity;

[0012] S212, tissue digestion: cut the tumor tissue into small pieces, digest it in a digestive enzyme solution, and filter and collect the cell suspension;

[0013] S213, cell culture: mix the transfected cell suspension with matrix gel, add it dropwise to the culture plate, and add organoid culture medium for culture after solidification;

[0014] S214, initial culture: culture in organoid culture medium for 7-14 days, during which the culture medium is replaced;

[0015] S215, subculture: subculture by mechanical or chemical methods, and collect for later use;

[0016] S216, organoid inoculation: Take out the prepared blank organ chip, inoculate the organoid sample mixed with matrix gel into the reserved chip hole / channel, and place it in a cell culture incubator to wait for the matrix gel to solidify;

[0017] S22, construction of vascularized microenvironment;

[0018] S221. Cell culture: Take frozen human vascular endothelial cells, resuscitate, resuspend and centrifuge them, transfer them into cell culture bottles, and place them in a cell culture incubator for standby use;

[0019] S222. Cell inoculation: Take out the organ chip inoculated with organoids, inoculate the human vascular endothelial cell suspension into the reserved chip holes / channels, and place it on a rocking perfusion instrument in a cell culture incubator for culture to ensure that the vascular endothelium forms a dense lumen;

[0020] S223. Model verification: Organ chip modules were randomly selected to verify the density of vascularized channels and characterize the barrier function, proving that the constructed vascularized lumen had good density and barrier function and could be used as a drug delivery pathway for drug testing;

[0021] S224. Vascular network: After the vascular lumen is dense, vascular growth factors are added into the channel to stimulate vascular endothelial cells to penetrate into the matrix gel mixed with organoid samples to construct an organoid vascularized microenvironment.

[0022] S3, using the vascularized co-culture organ chip constructed in step S2 to perform drug sensitivity testing of anti-tumor combined drug regimens;

[0023] S31. Group the test drugs;

[0024] S32, using organ chips for drug sensitivity testing;

[0025] S33. Conduct drug sensitivity assessment on organ chips.

[0026] S4. Tumor growth inhibiting drugs in the drug combination selected based on the results of drug sensitivity testing are used as alternative drugs with potential combined effects.

[0027] Furthermore, the drug regimen of the experimental group in S31 is a combination of an angiogenesis inhibitor and a tumor growth inhibitor. A blank group and a control group are set up at the same time. The dosage is designed in a gradient based on the maximum blood drug concentration Cmax value of the reference drug. The number of drug combinations to be tested is allocated based on the single-chip flux number of the organ chip and the number of repetitions of each experimental group.

[0028] Furthermore, S32 also includes:

[0029] S321. Before drug administration, perform preliminary high-content image recognition and fluorescence quantitative recognition on the constructed vascularized co-culture organ chip, including the seeded organoids and vascular networks;

[0030] S322. In accordance with the designed dosing sequence and dosing frequency, the drug regimen is injected into the reserved vascularized lumen channel, and after the drug injection is completed, the cell is placed on a rocking perfusion apparatus in a cell culture incubator for drug intervention culture;

[0031] S323. During the drug administration period, high-content image recognition is performed regularly on the organ chips cultured with drug intervention; at the end of drug administration, immunofluorescence staining and fluorescence quantitative identification are performed on the organ chips of all groups.

[0032] Furthermore, S33 also includes:

[0033] S331. Organoid area: Use a high-content imaging analyzer to take images of organoids, collect image data and analyze the area change ratio (OAC) of the organoids to determine drug sensitivity;

[0034] Endpoint OAC formula:

[0035] OAC 1 (%) = (1-ROAT / ROA C )*100%

[0036] ROA T =(T t -T 0 ) / T 0

[0037] ROA C =(C t -C 0 ) / C 0

[0038] T 0 : recognition area of ​​the experimental / control group organoids when administered; T t : The recognition area of ​​the experimental group / control group organoids at time t; C 0 : The recognition area of ​​the blank group organoids when administered; C t : The recognition area of ​​the blank group organoids at time t

[0039] According to the technical guidelines for nonclinical research of anticancer drugs, when OAC 1 When ≥60%, it is considered as drug sensitivity positive; when OAC 1 When the result is less than 60%, it is judged as drug sensitivity negative;

[0040] Autologous OAC formula:

[0041] OAC 2 (%) = (A t -A 0 ) / A 0 *100%

[0042] A0 : The recognition area of ​​each group of organoids when administered, A t : The identification area of ​​each group of organoids at time t;

[0043] According to the solid tumor efficacy evaluation criteria: when OAC 2 <20%, it is considered as drug sensitivity positive; when OAC 2 ≥20%, judged as drug sensitivity negative;

[0044] Comprehensive Assessment: When OAC 1 ≥ 60% and OAC 2 When the rate is less than 20%, the drug is considered effective. 1 <60% and OAC 2 When the rate is ≥20%, the drug is judged to be ineffective; other cases are judged to be potentially sensitive;

[0045] S332, Cell viability: Fluorescence image recognition of organoids and vascular networks was performed using a fluorescence microscope, and the degree of fluorescence color change was used to reflect the drug sensitivity, including apoptosis: FITC color; necrosis: PI color;

[0046] Fluorescence quantitative data were collected and analyzed for the increased rate of apoptosis (CAC) and the increased rate of necrosis (CNC). Increased apoptosis and necrosis mean weakened cell viability.

[0047] CAC formula:

[0048] CAC(%)=(CA t -CA c ) / OA c *100%

[0049] Among them, CA c is the FITC fluorescence value of the blank group at time t; CAt is the FITC fluorescence value of the experimental group / control at time t;

[0050] CNC formula:

[0051] CNC(%)=(CN t -CN c ) / CN c *100%

[0052] CN c is the PI fluorescence value of the blank group at time t; CN t : PI fluorescence value of experimental group / control group at time t;

[0053] The drug sensitivity of the drug regimen was comprehensively evaluated by combining the autologous OAC value, endpoint OAC value, CNC value, CAC value, etc. of the experimental group, and a comparative analysis was performed on the drug regimens of multiple experimental groups.

[0054] Furthermore, the analysis method in S332 is to perform pairwise comparison of the differences in drug sensitivity data between the test groups, and the comparison order is autologous OAC value, endpoint OAC value, CNC value, and CAC value;

[0055] All drug sensitivity data were statistically analyzed using SPSS software. For measurement data with homogeneous variance and normal distribution, two independent sample t-test was used for comparison between two groups, and one-way analysis of variance was used for three groups or more. Nonparametric tests and multiple comparisons were used for measurement data that did not meet the homogeneous variance or normal distribution. p < 0.05 was considered to be significantly different.

[0056] Based on the ranking results, the drug regimen of the experimental group that is superior to the control group is screened out, and the tumor growth inhibiting drugs in the drug combination can be used as alternative drugs with potential combination effects.

[0057] Compared with the prior art, the present invention has the following beneficial technical effects:

[0058] The present invention makes a preliminary discovery of potential candidate drugs based on open source data, and combines it with high-throughput detection of organ chips to form a systematic and efficient screening method; constructs a vascularized organ chip model, which can simultaneously have the advantages of simulating tumor microenvironment and high-throughput detection; at the same time, the discovery of drug sensitivity-related markers can provide a basis for precise drug treatment and improve the clinical effectiveness of drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Schematic diagram of the process of the method in the embodiment of the present invention;

[0060] Figure 2 Schematic diagram of the process of drug sensitivity test in the embodiment of the present invention;

[0061] Figure 3 Schematic diagram of the structure of the organ chip in an embodiment of the present invention. DETAILED DESCRIPTION

[0062] Example 1

[0063] The present invention proposes a method for screening anti-angiogenic combination drugs using vascularized co-cultured organ chips, and selects drugs for lung cancer indications by taking lung cancer as the indication object;

[0064] The first step is database screening

[0065] a) Select drugs for lung cancer indications (including angiogenesis inhibitors (1) and tumor growth inhibitors (2)) as candidate drug types. Collect drug sensitivity test data (IC50 values) for lung cancer-related cell lines from the GDSC database, and collect the maximum plasma drug concentration (Cmax value) of the corresponding drugs from the DrugBank database, and compare the two. When IC50 < Cmax, the drug is considered sensitive (Y); when IC50 > Cmax, the drug is considered insensitive (N).

[0066] b) Statistically analyze the drug sensitivity ratio of cell lines and the effectiveness rate of drugs, and screen drugs with an effectiveness rate greater than 50% as candidate drugs for secondary screening in vitro experiments. The results are shown in the following table:

[0067] Drug 1-1 Drugs 1-2 Drugs 1-3 Drug 2-1 Drug 2-2 …… Sensitive ratio Cell Line 1 Y N N Y Y …… 56％ Cell Line 2 N Y Y Y Y …… 97％ Cell Line 3 N Y N Y N …… 85％ Cell Line 4 Y N N N Y …… 34％ Cell Line 5 Y N N Y Y …… 79％ …… …… …… …… …… …… …… …… Efficiency 67％ 48％ 24％ 77％ 89％ ……

[0068] (2) In vitro screening of cell lines

[0069] a) Cell culture

[0070] 1) Selection of cell lines: Select appropriate lung cancer cell lines (drug sensitivity ratio greater than 50%) for experiments.

[0071] 2) Preparation of culture medium: Prepare DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin.

[0072] 3) Cell culture: Inoculate lung cancer cells into culture flasks and culture them in an incubator at 37°C and 5% CO2 until the cells reach 70 - 80% confluence.

[0073] b) Drug treatment

[0074] 1) Preparation of drug solution: Prepare a drug stock solution with DMSO or other suitable solvents according to the characteristics and usage requirements of the drug.

[0075] 2) Cell inoculation: After digesting and counting the cells, inoculate them into 96-well plates at an appropriate density (such as 5,000 - 10,000 cells / well), and add 100 μL of culture medium to each well.

[0076] 3) Drug dilution: Dilute the drug in a gradient in the 96-well plate, usually setting different concentration gradients (such as 0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM), and set at least three replicate wells for each concentration.

[0077] 4) Drug treatment: Add the drug solution to the cell wells to make the final volume 100 μL, and set up a blank control group (only add culture medium) and a solvent control group (add the corresponding concentration of solvent).

[0078] c) Cell culture

[0079] Specific operation: Drug treatment time: Place the drug-treated cells in an incubator and incubate for 24-72 hours. The specific time depends on the mechanism of action of the drug and the characteristics of the cell line.

[0080] d) Cell viability assay (CTG method)

[0081] 1) Prepare CellTiter-Glo reagent: According to the kit instructions, mix the CellTiter-Glo solution with the buffer and shake gently.

[0082] 2) Add reagents: After drug treatment, remove the 96-well plate and wait for the plate temperature to equilibrate to room temperature. Add an equal amount of CellTiter-Glo reaction reagent (usually 100 μL) to each well.

[0083] 3) Mixing: After adding the reagents, shake the 96-well plate for 2 minutes on an oscillator to ensure thorough mixing.

[0084] 4) Incubation: Incubate the 96-well plate at room temperature for 10 minutes to allow the reaction to complete and stabilize the luminescent signal.

[0085] 5) Luminescent signal detection: Use a microplate luminometer to detect the luminescent signal of each well and record the data.

[0086] e) Data analysis

[0087] 1) Data collation: The obtained luminescence signal data are collated and imported into analysis software (such as GraphPad Prism).

[0088] 2) Calculation of cell viability: Taking the luminescence signal of the blank control group as 100%, the relative cell viability of each drug-treated group was calculated.

[0089] 3) IC50 calculation: The half inhibitory concentration (IC50) of the drug on lung cancer cells was calculated by nonlinear regression curve fitting.

[0090] 4) Data comparison: Evaluate the antitumor activity of drugs against lung cancer cells based on IC50 values ​​and cell viability at different concentrations; sort by IC50 values ​​to screen out the top three drugs in the angiogenesis inhibition category and tumor growth inhibition category.

[0091] Step 2: Construction of vascularized co-culture organ-on-a-chip

[0092] The organ chip based on the present invention is a membrane-free barrier chip constructed using Phaseguide technology. The overall appearance is a 384-well plate. A chip plate contains 40 separate chips, each of which consists of three channels: upper, middle and lower. Its structure is as follows: Figure 3As shown, the specific structure has been disclosed in the Chinese patent publication number CN118421563A, and will not be repeated here.

[0093] (1) Organoid preparation and seeding

[0094] a) Sample collection: Collect multiple different human tumor tissue samples to maintain tissue activity;

[0095] b) Tissue digestion: Cut the tumor tissue into small pieces, digest it in a digestive enzyme solution, and filter and collect the cell suspension;

[0096] c) Cell culture: The transfected cell suspension was mixed with matrix gel and added dropwise to the culture plate. After solidification, organoid culture medium was added for culture.

[0097] d) Initial culture: Culture in organoid culture medium for 7-14 days, during which the culture medium is changed;

[0098] e) Subculture: subculture by mechanical or chemical methods and collect for future use;

[0099] f) Organoid inoculation: Take out the prepared blank organ chip, inoculate the organoid sample mixed with matrix gel into the reserved chip hole / channel, and place it in a cell culture incubator to wait for the matrix gel to solidify.

[0100] Specific operations:

[0101] 1) Take out the organoids from the CO2 incubator and move them to the operating table.

[0102] 2) Discard the culture medium in each well.

[0103] 3) Add an appropriate amount of 4°C pre-cooled PBS to each well and let stand for 1 min.

[0104] 4) Blow away the matrix gel in each well, then transfer the liquid in the well into a 15 mL centrifuge tube and centrifuge at 300 x g at 4°C for 5 min.

[0105] 5) After centrifugation, the tube is divided into three layers. Use a pipette to discard the middle and upper layers and keep the cell layer;

[0106] 6) Resuspend the cells in 1-2 mL of digestion solution, pipette and mix well, and place the centrifuge tube in a 37°C water bath for oscillation digestion for 1-3 minutes;

[0107] 7) After digestion is completed, determine whether digestion is complete under a microscope.

[0108] 8) Use a pipette to draw PBS three times the volume of the digestion solution, add it to the centrifuge tube, pipette and mix to stop the digestion, and centrifuge at 300xg, 4℃ for 5 minutes.

[0109] 9) After centrifugation, discard the supernatant, resuspend with 1 mL of PBS, and centrifuge at 300 x g, 4°C for 5 min.

[0110] 10) After centrifugation, discard the supernatant, resuspend the precipitate in matrix gel in an ice bath to an appropriate density, aspirate 1-2 μL, add to the B1 or B3 inoculation well, and then place in a carbon dioxide incubator and let stand for 20 minutes to allow the gel to solidify.

[0111] (2) Construction of vascularized microenvironment

[0112] a) Cell culture: Take frozen human vascular endothelial cells, resuscitate, resuspend, centrifuge, transfer into cell culture bottles, and place in a cell culture incubator for standby use;

[0113] b) Cell inoculation: Take out the organ chip inoculated with organoids, inoculate the human vascular endothelial cell suspension (mixed cell culture medium) into the reserved chip holes / channels, and place it on a rocking perfusion instrument in a cell culture incubator for culture to ensure that the vascular endothelium forms a dense lumen;

[0114] Specific operations:

[0115] 1) While waiting for the gel to solidify, collect endothelial cells (T25): discard the cell culture medium, wash each bottle of cells twice with PBS, then add 2 mL of digestion solution for digestion, and quickly add 2 mL of the corresponding culture medium of equal volume to terminate the digestion.

[0116] 2) After terminating the digestion, blow the cells with a pipette tip with appropriate force to blow the cells off the surface as completely as possible, then collect the liquid into a 15 mL centrifuge tube and centrifuge at 300xg for 5 minutes.

[0117] 3) After centrifugation, discard the supernatant and resuspend the endothelial cells in co-culture medium to a density of approximately 1x10 7 ~2x10 7 Pieces / mL.

[0118] 4) Take out the solidified chip plate, pipette 2 μL of endothelial cell suspension into well A1 or A3, and then tilt the chip 90° from A to C with channel B as the axis for about 1 hour to allow the cells to adhere to the matrix gel side, and then add culture medium (no more than 40 μL per well), and add an equal amount of culture medium to channel C.

[0119] 5) The chip is placed on a rocking perfusion instrument in an incubator for culture. The rocking direction is up and down (A to C and C to A), the angle is set to 7° to 10°, and the rocking period is 15 to 30 minutes.

[0120] 6) After the vascular endothelium of pathway A forms a dense lumen, it can be used for drug testing.

[0121] c) Model validation: Organ chip modules were randomly selected to verify the density of the vascularized channel and characterize the barrier function, proving that the constructed vascularized lumen had good density and barrier function and could be used as a drug delivery pathway for drug testing.

[0122] d) Vascular network: After the vascular lumen is dense, vascular growth factors are added into the channel to stimulate endothelial cells to penetrate into the matrix gel mixed with organoid samples to construct an organoid vascularized microenvironment.

[0123] Step 3: Drug sensitivity test for anti-tumor combined drug regimen

[0124] (1) Drug regimen grouping

[0125] a) The drug regimen of the experimental group is a combination of an angiogenesis inhibitor and a tumor growth inhibitor, and a blank group and a control group (an angiogenesis inhibitor alone group) are set up. The results are shown in the following table:

[0126]

[0127] b) The dosage is designed in a gradient based on the maximum blood drug concentration (Cmax value) of the drug, and the number of drug combinations to be tested is allocated based on the single-chip flux number of the organ chip and the number of repetitions in each test group.

[0128] Concentration gradient: 0.1Cmax, 0.3Cmax, Cmax, 3Cmax, 10Cmax

[0129] (2) Organ Chip Drug Sensitivity Test

[0130] a) Before drug administration, the constructed vascularized co-culture organ chip was preliminarily imaged with high content images, and a random portion was selected for initial fluorescence quantitative staining identification, including the seeded organoids and vascular networks;

[0131] The details are as follows:

[0132] 1) Use a high-content imaging analyzer to locate, photograph, and scan the inoculated organoids and vascular networks in all groups to collect initial imaging data; after photography, spray the surface with 75% alcohol to disinfect it and return it to the carbon dioxide incubator for culture.

[0133] 2) Randomly select some vascularized co-cultured organ chips, discard the culture medium, and wash with PBS three times.

[0134] 3) Add a mixture of FITC / PI:Binding Buffer at a volume ratio of 1:200 and incubate at room temperature for 25 minutes.

[0135] 4) Place the sample under a fluorescence microscope to observe the staining and collect initial imaging data.

[0136] b) According to the designed dosing sequence and frequency, the drug regimen is injected into the reserved vascularized lumen channel, and after the drug addition is completed, it is placed on a rocking perfusion instrument in a cell culture incubator for drug intervention culture;

[0137] Specific operations:

[0138] 1) Add 20 μL of drug-containing culture medium to the middle hole of the chip, and add 60 μL of drug-containing culture medium to channel A or channel C.

[0139] 2) Place the chip plate on a rocking perfusion instrument in a carbon dioxide incubator (angle set to 25°, rocking interval 15 min) for incubation, and replenish the drug on day 2, day 4, and day 6.

[0140] c) During the drug administration period, high-content image recognition was performed regularly on the organ chips cultured with drug intervention; at the end of drug administration, immunofluorescence staining (FITC / PI) and fluorescence quantitative identification were performed on the organ chips of all groups.

[0141] Specific operations:

[0142] 1) During the drug administration period, the inoculated organoids and vascular networks in all groups were positioned, photographed and scanned, and the area changes of the organoids were analyzed by imaging software.

[0143] 2) After reaching the dosing endpoint, the culture medium was discarded and the cells were washed three times with PBS.

[0144] 3) Add a mixture of FITC / PI:Binding Buffer at a volume ratio of 1:200 and incubate at room temperature for 25 minutes.

[0145] 4) Place the cells under a fluorescence microscope and observe the staining. Cells in the early stage of apoptosis are FITC positive, and cells in the late stage of apoptosis or necrosis are PI positive.

[0146] (3) Organ Chip Drug Sensitivity Assessment

[0147] Before and after the administration cycle, the changes in organoid area (OAC) and cell viability (CAC / CNC) were compared and analyzed. 1 ≥ 60% and OAC 2 When the rate is less than 20%, the drug is considered effective (P); when OAC 1 <60% and OAC 2 When the sensitivity is ≥20%, the drug is judged as ineffective (N); other cases are judged as intermediate sensitivity (M).

[0148] Take the drug sensitivity test of organoid 1 sample as an example:

[0149] <![CDATA[OAC 1 ]]> <![CDATA[OAC 2 ]]> CAC CNC Efficacy evaluation Combination 1-1 35％ 46％ 15％ 24％ N Combination 1-2 83％ -33％ 57％ 27％ P Combination 1-3 69％ 38％ 25％ 29％ M Drug 1-1 74％ -28％ 23％ 36％ P …… …… …… …… …… ……

[0150] Evaluation results: Drug combination 1-2 and control group drug 1-1 were confirmed to be effective, and drug combination 1-3 was intermediately sensitive.

[0151] Compare the differences between the drug sensitivity data of combination 1-2 and drug 1-1:

[0152] 1. Changes in organoid area (OAC) 1 , %)

[0153] Combination 1-2: a.81; b.79; c.89

[0154] Drug 1-1: a.78; b.69; c.75

[0155] Difference analysis: p>0.05

[0156] 2. Changes in organoid area (OAC) 2 , %)

[0157] Combination 1-2: a.-24; b.-34; c.-41

[0158] Drug 1-1: a.-27; b.-32; c.-25

[0159] Difference analysis: p>0.05

[0160] 3. Increased rate of organoid cell apoptosis (CAC, %)

[0161] Combination 1-2: a.59; b.51; c.61

[0162] Drug 1-1: a.20; b.25; c.24

[0163] Analysis of difference: p<0.05

[0164] Comprehensive evaluation: combination 1-2 is better than drug 1-1.

[0165] Detailed results for all organoid samples are shown below:

[0166]

[0167]

[0168] Comprehensive evaluation: Combination 1-2 has the highest absolute sensitivity.

[0169] The drug regimens in the experimental group with the highest absolute sensitivity rate that are superior to the control group are screened out, and the tumor growth-inhibiting drugs in their drug combinations can be used as alternative drugs with potential combined effects. Some drugs with significant effects are selected for repeated experiments to verify the reliability of the results.

[0170] Disadvantages of existing technologies: Monotherapy with angiogenesis-inhibiting drugs can easily lead to drug resistance in tumor cells, resulting in reduced efficacy. Although combination therapy can improve efficacy, if reasonable screening cannot be achieved in combination therapy, it is often accompanied by more complex drug resistance risks and treatment responses; traditional two-dimensional cell culture and animal models cannot fully simulate the microenvironment of human tumors, resulting in insufficient accuracy in drug screening and evaluation. The medication regimen after screening lacks a precise basis for guiding medication. After adopting this scheme, potential candidate drugs can be preliminarily discovered based on open source data, combined with high-throughput detection of organ chips, to form a systematic and efficient screening method; constructing a vascularized organ chip model can have the advantages of simulating tumor microenvironment and high-throughput detection at the same time. The discovery of drug sensitivity-related markers can provide a basis for precise drug treatment and improve the clinical effectiveness of drugs.

[0171] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited thereto, and various changes can be made within the knowledge scope of technicians in the relevant technical field without departing from the purpose of the present invention.

Claims

1. A method for screening anti-angiogenic combination drugs using vascularized co-cultured organ chips, characterized in that: The specific steps include: S1. Screen candidate drugs with potential sensitivity to indications based on open source databases and perform in vitro cell screening; S2. Construction of vascularized co-culture organ chips; S3, using the vascularized co-culture organ chip constructed in step S2 to perform drug sensitivity testing of anti-tumor combined drug regimens; S4. Tumor growth inhibiting drugs in the drug combination selected based on the results of drug sensitivity testing are used as alternative drugs with potential combined effects.

2. The method for screening anti-angiogenic combination drugs using vascularized co-cultured organ chips according to claim 1, characterized in that: The types of indications targeted in S1 include lung cancer, colorectal cancer, liver cancer, pancreatic cancer or gastric cancer.

3. The method for screening anti-angiogenic combination drugs using vascularized co-cultured organ chips according to claim 2, characterized in that: S1 collects drugs in open-source databases that have completed indication-related cell lines, including IC50 values ​​of sensitivity test data for angiogenesis inhibition and tumor growth inhibition and Cmax values ​​of blood drug concentration data of corresponding drugs, and compares the two; The drug sensitivity test data comes from the GDSC database, and the maximum blood drug concentration Cmax value comes from the DrugBank database.

4. The method for screening anti-angiogenic combination drugs using vascularized co-cultured organ chips according to claim 3, characterized in that: In S1, in vitro cell line screening is performed on the candidate drugs screened out from the database to obtain the IC50 values ​​of the sensitivity test data of drugs including those inhibiting angiogenesis and inhibiting tumor growth, and compare them with the Cmax values ​​of the corresponding blood drug concentration data to verify the accuracy of the database screening and further screen the candidate drugs to eliminate insensitive drugs.

5. The method for screening anti-angiogenic combination drugs using vascularized co-cultured organ chips according to claim 1 or 4, characterized in that: S2 also includes: S21, Organoid preparation and seeding; S211. Sample collection: Collect multiple different human tumor tissue samples to maintain tissue activity; S212, tissue digestion: cut the tumor tissue into small pieces, digest it in a digestive enzyme solution, and filter and collect the cell suspension; S213, cell culture: mix the transfected cell suspension with matrix gel, add it dropwise to the culture plate, and add organoid culture medium for culture after solidification; S214, initial culture: culture in organoid culture medium for 7-14 days, during which the culture medium is replaced; S215, subculture: subculture by mechanical or chemical methods, and collect for later use; S216, organoid inoculation: Take out the prepared blank organ chip, inoculate the organoid sample mixed with matrix gel into the reserved chip hole / channel, and place it in a cell culture incubator to wait for the matrix gel to solidify; S22, construction of vascularized microenvironment; S221. Cell culture: Take frozen human vascular endothelial cells, resuscitate, resuspend and centrifuge them, transfer them into cell culture bottles, and place them in a cell culture incubator for standby use; S222. Cell inoculation: Take out the organ chip inoculated with organoids, inoculate the human vascular endothelial cell suspension into the reserved chip holes / channels, and place it on a rocking perfusion instrument in a cell culture incubator for culture to ensure that the vascular endothelium forms a dense lumen; S223. Model verification: Organ chip modules were randomly selected to verify the density of vascularized channels and characterize the barrier function, proving that the constructed vascularized lumen had good density and barrier function and could be used as a drug delivery pathway for drug testing; S224. Vascular network: After the vascular lumen is dense, vascular growth factors are added into the channel to stimulate vascular endothelial cells to penetrate into the matrix gel mixed with organoid samples to construct an organoid vascularized microenvironment.

6. The method for screening anti-angiogenic combination drugs using vascularized co-cultured organ chips according to claim 5, characterized in that: S3 also includes: S31. Group the test drugs; S32, using organ chips for drug sensitivity testing; S33. Conduct drug sensitivity assessment on organ chips.

7. The method for screening anti-angiogenic combination drugs using vascularized co-cultured organ chips according to claim 6, characterized in that: The drug regimen of the experimental group in S31 is a combination of an anti-angiogenesis drug and a tumor growth inhibitory drug. A blank group and a control group are set up at the same time. The dosage is designed in a gradient based on the maximum blood drug concentration Cmax value of the reference drug. The number of drug combinations to be tested is allocated based on the single-chip flux number of the organ chip and the number of repetitions in each experimental group.

8. The method for screening anti-angiogenic combination drugs using vascularized co-cultured organ chips according to claim 6, characterized in that: The S32 also includes: S321. Before drug administration, perform preliminary high-content image recognition and fluorescence quantitative recognition on the constructed vascularized co-culture organ chip, including the seeded organoids and vascular networks; S322. In accordance with the designed dosing sequence and dosing frequency, the drug regimen is injected into the reserved vascularized lumen channel, and after the drug injection is completed, the cell is placed on a rocking perfusion apparatus in a cell culture incubator for drug intervention culture; S323. During the drug administration period, high-content image recognition is performed regularly on the organ chips cultured with drug intervention; at the end of drug administration, immunofluorescence staining and fluorescence quantitative identification are performed on the organ chips of all groups.

9. The method for screening anti-angiogenic combination drugs using vascularized co-cultured organ chips according to claim 6, characterized in that: S33 also includes: S331. Organoid area: Use a high-content imaging analyzer to take images of organoids, collect image data and analyze the area change ratio (OAC) of the organoids to determine drug sensitivity; Endpoint OAC formula: OAC1(%)=(1-ROAT / ROA C )*100% LONG T =(T t -T0) / T0 ROA C =(C t -C0) / C0 T0: identification area of ​​the experimental / control group organoids when the drug was administered; T t : recognition area of ​​the organoids in the experimental group / control group at time t; C0: recognition area of ​​the organoids in the blank group when the drug was administered; C t : When the recognition area of ​​the blank group organoid at time t is OAC1 ≥ 60%, it is judged as drug sensitivity positive; when OAC1 < 60%, it is judged as drug sensitivity negative; Autologous OAC formula: OAC2(%)=(A t -A0) / A0*100% A0: identification area of ​​each group of organoids when administered, A t : The identification area of ​​each group of organoids at time t; When OAC2 is less than 20%, it is judged as drug sensitivity positive; when OAC2 is greater than or equal to 20%, it is judged as drug sensitivity negative; Comprehensive evaluation: When OAC1 ≥ 60% and OAC2 < 20%, the drug is judged to be effective; when OAC1 < 60% and OAC2 ≥ 20%, the drug is judged to be ineffective; other situations are judged as potential sensitivity; S332, Cell viability: Fluorescence image recognition of organoids and vascular networks was performed using a fluorescence microscope, and the degree of fluorescence color change was used to reflect the drug sensitivity, including apoptosis: FITC color; necrosis: PI color; Fluorescence quantitative data were collected and analyzed for the increased rate of apoptosis (CAC) and the increased rate of cell necrosis (CNC). Increased apoptosis and increased cell necrosis mean weakened cell viability. CAC formula: CAC(%)=(CA t -THAT c ) / OA c *100% Among them, CA c is the FITC fluorescence value of the blank group at time t; CAt is the FITC fluorescence value of the experimental group / control at time t; CNC formula: CNC(%)=(CN t -CN c ) / CN c *100% CN c is the PI fluorescence value of the blank group at time t; CN t : PI fluorescence value of experimental group / control group at time t; The drug sensitivity of the drug regimen was comprehensively evaluated by combining the autologous OAC value, endpoint OAC value, CNC value, CAC value, etc. of the experimental group, and a comparative analysis was performed on the drug regimens of multiple experimental groups.

10. The method for screening anti-angiogenic combination drugs using vascularized co-cultured organ chips according to claim 9, characterized in that: The analysis method in S332 was to conduct pairwise comparisons of differences in drug sensitivity data between the test groups, and the comparison order was autologous OAC value, endpoint OAC value, CNC value, and CAC value; All drug sensitivity data were statistically analyzed using SPSS software. For measurement data with homogeneous variance and normal distribution, two independent sample t-test was used for comparison between two groups, and one-way analysis of variance was used for three groups or more. Nonparametric tests and multiple comparisons were used for measurement data that did not meet the homogeneous variance or normal distribution. p < 0.05 was considered to be significantly different. Based on the ranking results, the drug regimen of the experimental group that is superior to the control group is screened out, and the tumor growth inhibiting drugs in the drug combination can be used as alternative drugs with potential combination effects.

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