Drug screening method based on liver cancer organoid
Through the drug screening method based on liver cancer organoids, the problem of lack of repeatable human models in the prior art has been solved, the development of personalized treatment of liver cancer has been achieved, and the accuracy and credibility of drug screening have been improved.
Patent Information
- Application Number
- CN202510366417.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-25
- Publication Date
- 2025-06-13
AI Technical Summary
The lack of reproducible human models in the prior art to evaluate the efficacy of candidate therapies limits the development of personalized treatment of liver cancer.
Provide a drug screening method based on liver cancer organoids. By preparing liver cancer organoids prepared from liver cancer tissues of different sources, obtaining pathological characteristics and genetic characteristics, conducting analysis and culture, adding candidate drugs to process, detecting cell viability and shape characteristic parameters, calculating IC50 values, constructing a candidate drug database, and determining candidate drugs for treatment.
It improves the accuracy and credibility of drug screening, and can determine suitable candidates based on the patient's own conditions and the development stage of liver cancer, as a personalized treatment tool for liver cancer patients.
Smart Images
Figure CN120138100A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the technical field of tumor organoids, and particularly to a drug screening method based on liver cancer organoids. Background Art
[0002] Liver cancer ranks fourth among various lethal cancers and is the sixth most common cancer disease in China. The incidence of liver cancer in China ranks among the top in the world, and the prognosis of patients is poor and the fatality rate is high. The treatment of liver cancer generally mainly combines surgical resection and chemotherapy. Most patients have already been in the middle and late stages of liver cancer when their diseases are discovered, and the treatment effect is not ideal. Moreover, most liver cancer patients are not sensitive to drug treatment, and there is an urgent need to develop personalized treatment methods. The traditional liver cancer research models mainly include in vitro cell line culture and animal models. The liver cancer cell lines cultured in vitro often lack liver-specific genes and specific biological functions, such as the maintenance of cytochrome P450, specific connections between cells, etc., and lack the interaction between different types of cells and between cells and the extracellular matrix, and cannot well reproduce the heterogeneity and complex structural characteristics of liver cells. Animal models have species differences, which limit the continuity of their clinical research, resulting in many research results that cannot be directly applied to clinical practice.
[0003] Therefore, the development of personalized treatment for liver cancer is restricted due to the lack of a reproducible human model to evaluate the efficacy of candidate treatments. Summary of the Invention
[0004] To solve the problems in the prior art, this application provides a drug screening method based on liver cancer organoids.
[0005] This application provides a drug screening method based on liver cancer organoids, adopting the following technical solutions:
[0006] A drug screening method based on liver cancer organoids includes the following steps:
[0007] S1, preparing liver cancer organoids from liver cancer tissues of different sources, wherein the liver cancer organoids prepared from liver cancer tissues of the same source are recorded as a group;
[0008] S2, obtaining pathological features and genetic features from the liver cancer organoids obtained in S1, analyzing the pathological features and the genetic features, and determining that the liver cancer organoids can correspond to the pathological features and the genetic features of the liver cancer tissues of the same source;
[0009] S3, culturing the liver cancer organoids analyzed by the pathological features and the genetic features in S2, then adding a candidate drug for treatment, and detecting cell viability and the shape-based characteristic parameters of the liver cancer organoids;
[0010] S4. Calculate the IC50 value according to the cell viability detection results. Each of the liver cancer tissues of the same origin corresponds to an IC50 value and a morphological characteristic parameter of the organoid. Use the IC50 values corresponding to the liver cancer tissues of different origins and the morphological characteristic parameters of the organoids to obtain a candidate drug database based on liver cancer organoids.
[0011] S5. Use the candidate drug database based on liver cancer organoids to determine candidate drugs for treatment.
[0012] The IC50 value is the half-inhibitory concentration, or half-inhibition rate, which refers to the concentration of a certain drug or substance when it inhibits certain biological processes (such as enzymes, cell receptors, or microorganisms) to achieve a 50% inhibitory effect. The smaller the IC50 value, the better the inhibitory effect of a certain drug or substance on certain biological processes.
[0013] The drug screening method based on liver cancer organoids provided by the present application uses liver cancer organoids prepared from liver cancer tumor tissues for anti-cancer drug screening, tests the cell activity and the shape-based characteristic parameters of liver cancer organoids after the action of candidate drugs, uses the IC50 value and the shape-based characteristic parameters of liver cancer organoids as the basis for determining the sensitivity of candidate drugs for liver cancer organoids from different patients, constructs a database covering the sensitive reactions of candidate drugs for liver cancer organoids from different patients, and each liver cancer tissue of a certain origin corresponds to an IC50 value and the shape-based characteristic parameters of liver cancer organoids. And each liver cancer tissue of a certain origin corresponds to a patient's own conditions and the corresponding stage of liver cancer development. Subsequently, according to the patient's own conditions and the corresponding stage of liver cancer development, the corresponding candidate drugs with the smallest IC50 value, few live cells in the liver cancer organoids, and no obvious cavity structure are determined from the above database, which can be used as a powerful tool for drug screening and personalized treatment of liver cancer patients.
[0014] Preferably, the pathological features include that the cell nuclei of the liver cancer organoids show large nuclei and dense chromatin after HE staining; the genetic features include at least one of OPN (cholangiocyte marker), AFP (liver cancer cell marker), CEACAM6 (liver cancer cell marker), and α-SMA (fibroblast marker).
[0015] Through the comparison of the above pathological features and genetic features, the present application confirms that the prepared liver cancer organoids can replace liver cancer tissues for drug screening, improving the accuracy and credibility of drug screening.
[0016] Preferably, the morphological characteristic parameters of the organoids include at least one of false glandular ring structures or glandular cavity structures.
[0017] Preferably, the overall structure of the liver cancer organoids dissociates and collapses, the pseudo-glandular striation structure or the glandular cavity-like structure disappears, and the number of living cells in the liver cancer organoids decreases.
[0018] Preferably, S1 includes the following steps:
[0019] S11, dissociating and digesting liver cancer tissues from different sources into single cells and mixing them with Matrigel to obtain a cell-Matrigel mixture;
[0020] S12, solidifying the cell-Matrigel mixture obtained in S11 to obtain cell microspheres;
[0021] S13, inoculating the cell microspheres obtained in S12 into a liver cancer organoid culture medium for culture to obtain liver cancer organoids.
[0022] In this application, liver cancer tissues are prepared into liver cancer organoids through microfluidic technology. Under the same number of cells and time, the speed of constructing liver cancer organoids in this application (construction time: 7 - 15 days) is significantly higher than that of constructing organoids in the prior art (construction time: 30 - 60 days), which helps to achieve the high-efficiency and high-throughput preparation of liver cancer organoids. Moreover, since the organoids in this application have a wide range of sources, they can be used as a basis for determining the homology of liver cancer tissues according to the patient's own conditions, cancer development stage, etc., thereby constructing a database with a wide range of data bases, providing a basis for the screening of candidate drugs for patients with different own conditions and different liver cancer stages in the future.
[0023] Preferably, the liver cancer tumor tissue in S11 is a human-derived liver cancer tumor tissue.
[0024] By using human-derived liver cancer tumor tissues to prepare liver cancer organoids, the formed liver cancer organoids retain the morphology and characteristics of the parental tissues, express liver cancer-related cell markers and liver cancer markers, improve the accuracy of later drug sensitivity tests, improve the accuracy of the data in the database, and provide a guarantee for the later screening of post-marketing drugs for patients based on this database.
[0025] Preferably, during the mixing process of S11, every 5×10 4 -10×10 4 single cells are mixed with 1 μL of the Matrigel.
[0026] In the method for preparing liver cancer organoids in this application, liver cancer organoids with uniform size can still be obtained while using less Matrigel.
[0027] Preferably, the flow rate of the cell-Matrigel mixture is 10 - 15 μL / min, and the flow rate of the fluorinated oil is controlled to be 100 - 110 μL / min.
[0028] Preferably, the particle size of the liver cancer organoids obtained in S14 is 450 - 550 μm.
[0029] The morphology and structure of the liver cancer organoids prepared in this application are uniform, which is beneficial to improving the accuracy of subsequent drug sensitivity tests of candidate drugs acting on liver cancer organoids and enhancing the credibility of the drug screening database based on liver cancer organoids.
[0030] Preferably, after the cell microspheres in S14 are inoculated into the liver cancer organoid culture medium, they are cultured in an environment of 37°C - 38.5°C and 5% - 7.5% CO 2 for 7 - 15 days. Description of the Drawings
[0031] Figure 1 The observation effect diagrams under the microscope of human-derived liver cancer organoids corresponding to 12 liver cancer patients.
[0032] Figure 2 The HE staining effect diagrams of human-derived liver cancer parental tissue (Figure a) and the corresponding human-derived liver cancer organoids (Figure b).
[0033] Figure 3 IF detection of intestinal epithelial cells, liver cancer cells and immune cell markers in human-derived liver cancer parental tissue and its organoids.
[0034] Figure 4 The candidate drug concentration-effect curves of human-derived liver cancer organoids after being treated with candidate drugs (the liver cancer organoids used in Figures a1, a2, a3 correspond to the T1b stage, T2 stage, T3 stage of the liver cancer tissue, and the candidate drug used is cisplatin; the liver cancer organoids used in Figures b1, b2, b3 correspond to the T1b stage, T2 stage, T3 stage of the liver cancer tissue, and the candidate drug used is lenvatinib; the liver cancer organoids used in Figures c1, c2, c3 correspond to the T1b stage, T2 stage, T3 stage of the liver cancer tissue, and the candidate drug used is anti PD-1).
[0035] Figure 5 The morphological characteristics of human-derived liver cancer organoids after being treated with candidate drugs (the liver cancer organoids used in Figures a1, a2, a3 correspond to the T1b stage, T2 stage, T3 stage of the liver cancer tissue, and the candidate drug used is cisplatin; the liver cancer organoids used in Figures b1, b2, b3 correspond to the T1b stage, T2 stage, T3 stage of the liver cancer tissue, and the candidate drug used is lenvatinib; the liver cancer organoids used in Figures c1, c2, c3 correspond to the T1b stage, T2 stage, T3 stage of the liver cancer tissue, and the candidate drug used is anti PD-1). Detailed Embodiments
[0036] For better understanding and implementation, the technical solutions of the present application will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all of the embodiments.
[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the specification of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application.
[0038] Unless otherwise stated, all numerical values representing amounts of ingredients, reaction conditions, etc. used in the specification and claims are understood to be modified by the term "about". Therefore, unless otherwise indicated, the numerical parameters set forth herein are approximate values that can vary depending on the desired properties to be obtained.
[0039] As used herein, "and / or" refers to one or all of the recited elements.
[0040] As used herein, "comprising" and "including" cover cases where only the recited elements are present and cases where there are other unrecited elements in addition to the recited elements.
[0041] All percentages in this application are weight percentages, unless otherwise specified.
[0042] Unless otherwise stated, the articles "a", "an", "the" and "said" used in this specification are intended to include "at least one" or "one or more". For example, "a component" refers to one or more components, and thus more than one component may be contemplated and may be employed or used in the implementation of the described embodiments.
[0043] Example 1
[0044] This example presents a drug screening method based on liver cancer organoids:
[0045] First step: Construction of liver cancer organoids
[0046] The preparation method of liver cancer organoids includes the following steps:
[0047] Tissue treatment and digestion: Take 12 human liver cancer tissues and place them in 6 cm culture dishes respectively. Number the 12 human liver cancer tissues as P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12. The specific information of the patients with the 12 human liver cancer tissues is shown in Table 1;
[0048] Table 1
[0049]
[0050] Aspirate the tissue preservation solution, add 3 mL of PBS to resuspend the tissue, and use sterilized surgical scissors and forceps to remove debris such as fat, smooth muscle layer, and blood stains on the tissue surface. Retain the crypt structure containing a large number of stem cells, and then wash the tissue 3 times with 1 - 3 mL of PBS until the washing solution is clear, and remove the washing solution. Resuspend the tissue with 1 - 3 mL of PBS containing 1% PSA. Add 5 mL of PBS containing 1% PSA to each well of a six - well cell culture plate. Hold the tissue with forceps and slowly rotate it clockwise 5 times in the well, and repeat the same operation for each well. Take a new 6 - cm culture dish, transfer the washed tissue to the culture dish, add 3 mL of PBS containing 1% PSA to resuspend the tissue, cut it into 2 - mm fragments, and use a pipette or Pasteur pipette to transfer the tissue and suspension to a 15 - mL centrifuge tube, and remove the PBS. Prepare the digestion solution: Dissolve 10 mg of collagenase, 10 μL of DNase, 10 μL of dispase in 10 mL of DMEM / F12 medium containing 1% BSA, prepare it immediately before use, and filter it through a 22 - μm filter membrane; add 5 mL of the digestion solution to the centrifuge tube containing the tissue fragments, seal the tube cap with a sealing film, place it in a shaker at 37 °C, place it obliquely, and incubate at 200 rpm for 15 - 30 min. Observe the dissociation state of the crypts every 5 min until it meets the dissociation standard, and add 2.5 mL (half of the total volume of the digestion solution) of medium containing 10% serum to terminate the digestion. Centrifuge the obtained tissue suspension at 300 g for 10 min at 4 °C, discard the supernatant, and add 5 mL of PBS containing 1% PSA to resuspend the precipitate. Place a 100 - μm cell sieve on a 50 - mL centrifuge tube with forceps, add 2 mL of PBS containing 1% PSA to rinse the sieve, transfer the resuspended tissue fluid to the cell sieve with a Pasteur pipette for filtration, gently tap the outer wall of the test tube to make the filtrate flow down slowly, wash the inner wall of the centrifuge tube used for digestion and the filter residue on the sieve twice with an appropriate amount of PBS containing 1% PSA, and examine the filtrate under a microscope to observe the number of crypts. Centrifuge the collected tissue filtrate at 300 g for 10 min at 4 °C, discard the supernatant, observe the number of red blood cells, add 1 - 3 mL of red blood cell lysate, let it stand at room temperature for 3 min for lysis, add an equal volume of PBS containing 1% PSA to terminate the red blood cell lysis reaction, centrifuge in a centrifuge at 300 g for 5 min at 4 °C, and discard the supernatant. Add 1 mL of complete culture medium to resuspend the cell precipitate. Take 10 μL of the cell suspension and add it to 10 μL of AOPI (Acid Orange 7), mix well, and inject 20 μL of the mixed solution onto a clean counting plate for cell counting.
[0051] Cell microsphere preparation: Place sterilized 100 - μL pipette tips in a - 20 °C refrigerator for pre - cooling 1 day in advance. According to the cell counting results, at 1×10 5The volume of Matrigel required for cell / μL concentration calculation. Add Matrigel to the cell pellet with a pre-chilled pipette tip, gently stir several times on ice and then pipette up and down to ensure no air bubbles are generated. After mixing until there are no cell clumps, transfer the resulting cell-Matrigel mixture to a 96-well plate, 10-20 μL per well, and place it in an incubator at 37 °C for 15 min to obtain cell microspheres.
[0052] Preparation of human liver cancer organoids: Pipette the cell microspheres into a petri dish containing human liver cancer medium with a 10 mL syringe, and gently aspirate the fluorinated oil on the surface of the petri dish with a 1 mL syringe with a needle. Place it in an incubator at 37 °C with 5.0% CO 2 and culture for 7 days. Replace the fresh medium on the first day, observe and take pictures under bright field microscopy every day, and record whether organoid structures grow. After culturing for 7 days, the liver cancer organoid model matures, and obvious glandular cavity-like structures appear in the sphere (such as Figure 1 ).
[0053] Among them, the medium used in the process of organoid preparation is prepared according to the following formula: Advanced DMEM / F12 supplemented with 1% penicillin / streptomycin, 1% glutamax, 10-mM HEPES, 1:50 B27 (without vitamin A), 1:100 N2, 1.25 mM N-acetyl-L-cysteine, 10-mM nicotinamide, 10-nM recombinant human (Leu15)-gastrin I, 50 ng / mL recombinant human EGF, 100 ng / mL recombinant human FGF10, 25 ng / mL recombinant human HGF, 10 mM forskolin, 5-mM A83-01, 10 mM Y27632
[0054] 25 ng / mL recombinant human Noggin, 500 ng / mL Rspo-1 and 100 ng / mL Wnt3a.
[0055] Analysis 1: HE staining of human liver cancer organoids:
[0056] Perform HE staining on the above-prepared human liver cancer organoids, specifically including:
[0057] Step 1) Paraffin embedding of organoids
[0058] Preheat the embedding machine to 65 °C in advance.
[0059] Fixation: Add 4% PFA (about 200 μL) with a volume 10 times that of the organoids to an EP tube and fix at room temperature for 20 min. Pre-staining of organoids: Carefully aspirate the PFA with a 100 μL pipette, add 20 μL of eosin solution, and stain at room temperature for 5 min;
[0060] Organoid dehydration: Add 10 times the volume of 75% ethanol and dehydrate at room temperature; aspirate the 75% ethanol, add 10 times the volume of 90% ethanol, and dehydrate at room temperature for 5 min;
[0061] Organoid counterstaining: Add 20 μL of eosin solution and stain at room temperature for 5 min. (Counterstaining can be performed if the decolorization is severe);
[0062] Organoid dehydration: Add 10 times the volume of absolute ethanol and dehydrate at room temperature for 5 min, repeat three times;
[0063] Organoid clearing: Add 10 times the volume of xylene and soak at room temperature for 5 min, repeat three times;
[0064] Organoid embedding in wax: Take a white embedding cassette, remove the lid, and label the organoid batch number on the cassette. Wait for the organoids to sink to the bottom, carefully aspirate the upper layer of xylene (leave 50 μL), use a 1 mL pipette tip with the tip cut off to aspirate the sample, transfer the sample to a paraffin mold, add paraffin, and soak at 65 °C for 10 min; tilt the mold to discard the paraffin, add paraffin again and soak for 10 min, gently place the white embedding cassette on the paraffin mold, and let it stand at 65 °C for 10 min. After the paraffin solidifies, store the wax block at room temperature.
[0065] Step 2) Sectioning and HE staining
[0066] The paraffin-embedded block is sectioned on a microtome with a thickness of 5 μm;
[0067] Section dewaxing to water: Sequentially place the sections in xylene I for 5 min, xylene II for 5 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, 95% ethanol for 5 min, 90% ethanol for 5 min, 80% ethanol for 5 min, 70% ethanol for 5 min, and wash with distilled water;
[0068] Hematoxylin staining of cell nuclei: Preheat the water bath to 60 °C in advance, soak the sections in hematoxylin and stain at 60 °C for 3 - 5 min, soak in tap water for a while to remove the excess hematoxylin, differentiate with 1% acidic ethanol differentiation solution for 20 s, soak and wash with tap water, blue with 1% ammonia water for 20 s, and soak and wash with tap water;
[0069] Eosin staining of cytoplasm: Soak the sections in eosin staining solution and stain for 30 s;
[0070] Dehydration and sealing: Put the slices in 95% alcohol I for 5 minutes, 95% alcohol II for 5 minutes, anhydrous ethanol I for 5 minutes, anhydrous ethanol II for 5 minutes, xylene I for 5 minutes, and xylene II for 5 minutes to dehydrate and make them transparent. Take the slices out of xylene, put a clean absorbent paper on the table, take the slide out of xylene and put it on the paper (with the slice side facing up), quickly drop a drop of gum in the center of the slice, hold the right side of the cover glass gently with tweezers, slightly tilt it so that its left side touches the sealing medium, and then slowly put the cover glass down to reduce or avoid bubbles. Then perform microscopic examination and image acquisition and analysis.
[0071] Most liver cancers are classified into adenocarcinoma, undifferentiated carcinoma, adenosquamous carcinoma, small cell carcinoma and carcinoid according to their histological types, of which adenocarcinoma accounts for more than 95% of liver cancers. HE staining results showed that liver cancer organoids highly reproduced the histological characteristics of the parental tumor, showing cystic structures or dense structures without lumens, with large nuclei and dense chromatin (such as Figure 2 ).
[0072] Analysis 2: IF detection of human liver cancer organoids:
[0073] The human liver cancer organoids prepared above were subjected to IF testing, specifically including:
[0074] Step 1) Organoid paraffin embedding
[0075] The specific steps are the same as step 1 in Example 2)
[0076] Step 2) Slice IF detection
[0077] The paraffin sections were placed in a 65°C oven for 30 min-1 h until the wax dissolved.
[0078] Dewax paraffin sections to water: sequentially place the sections in xylene I for 20 min, xylene II for 10 min, xylene III for 10 min, anhydrous ethanol for 10 min, 95% ethanol for 10 min, 70% ethanol for 10 min, and wash with distilled water for 5 min;
[0079] Antigen repair: Place the slides in a repair box filled with EDTA antigen repair buffer (PH8.0) in a microwave oven for antigen repair. Heat on high heat for 5 minutes, medium heat for 20 minutes, and then switch to low heat for 2 minutes. During this process, prevent the buffer from evaporating excessively and do not dry the slides. After cooling naturally, place the slides in PBS (PH7.4) and wash them twice on a decolorizing shaker, 5 minutes each time.
[0080] Blocking: Add 100 μL of 5% BSA, 0.3% Triton X-100 to each section for 1 hour;
[0081] Primary antibody incubation: Dilute the primary antibody with the blocking solution to an appropriate concentration, add 50 μL of the primary antibody to each slice, and incubate overnight at 4°C;
[0082] Secondary antibody incubation: Take out the sections from the refrigerator and let them warm up for 20 - 30 min, wash 3 times with PBST, 10 min each time. Dilute the fluorescently labeled secondary antibody with PBS, add 50 μL of the secondary antibody to each slice, and incubate at room temperature for half an hour;
[0083] Counterstaining with DAPI: Wash 3 times with PBST, 10 min each time. Add the diluted DAPI and incubate at room temperature for 10 min, then wash with PBS for 5 min.
[0084] Mounting and microscopic examination: Add about 30 μL of anti - fluorescence quenching agent to each slice, cover with a coverslip, observe under a fluorescence microscope, and take pictures for record.
[0085] IF was used to detect the expression of hepatocyte markers, liver cancer markers, and immune cell markers in the parental liver cancer tissue and its organoids. IF was used to detect the expression of liver - related cell markers and liver cancer markers in the parental liver cancer tissue and its organoids. The results showed that both the parental tissue and the corresponding liver cancer organoids expressed OPN (cholangiocyte marker), AFP (liver cancer cell marker); expressed CEACAM6 (liver cancer cell marker) and α - SMA (fibroblast marker); indicating that the organoids restored the genetic characteristics of cancer cells in the tumor tissue (such as Figure 3 ).
[0086] The second step: Drug screening method based on liver cancer organoids
[0087] Seed the human liver cancer organoids with formed structures into a 96 - well plate protected from light, add 100 μL of medium containing different concentrations of candidate drugs to each well, and end the culture on the 6th day. Use the CellTiter - Glo (CTG) Luminescent Cell Viability Assay kit to detect the viability of tumor cell 3D microspheres. Thaw the CTG buffer at room temperature in advance and balance the temperature of the CTG substrate to room temperature; mix the CTG buffer and CTG substrate in a light - protected bottle, gently vortex to mix evenly to form CTG reagent; balance the temperature of the opaque 96 - well plate at room temperature for 30 min; add 100 μL of CTG reagent to each well and place it on a shaker and shake for 2 min to lyse the cells; incubate at room temperature for 10 min, after stabilizing the light signal, record the LUM light signal intensity with an enzyme - linked immunosorbent assay reader, make an IC 50 curve, read the IC50 values corresponding to different candidate drugs, and obtain a candidate drug database based on different liver cancer organoids (such as Figure 4 ).
[0088] Detecting the morphological characteristic parameters of organoids after the action of candidate drugs: Hepatocellular carcinoma organoids derived from T1b-stage, T2-stage, and T3-stage tissues showed a decrease in viable cells and no obvious lumen-like structures after treatment with cisplatin and lenvatinib. For hepatocellular carcinoma organoids derived from T1b-stage and T3-stage tissues, after treatment with anti-PD-1, there was no obvious change in the morphological structure of the hepatocellular carcinoma organoids, and they still had obvious organoid vesicular structures (such as Figure 5 ).
[0089] Therefore, for hepatocellular carcinoma tissues at the T1b stage, the candidate drugs should be cisplatin or lenvatinib; for hepatocellular carcinoma tissues at the T2 stage, the candidate drugs should be cisplatin, lenvatinib, or anti-PD-1; for hepatocellular carcinoma tissues at the T3 stage, the candidate drugs should be cisplatin or lenvatinib.
[0090] Subsequently, according to the individual conditions and cancer development stages of different patients, the candidate drugs suitable for the patients are determined from the above database based on the drug IC50 value and organoid morphology, and the candidate drugs are used to treat the corresponding hepatocellular carcinoma patients.
[0091] The above embodiments are only used to illustrate the technical solutions of the present application rather than to limit the protection scope of the present application. Although the present application has been described in detail with reference to the above embodiments, those of ordinary skill in the art should understand that the technical solutions of the present application can be modified or equivalently replaced, but these modifications or replacements are all within the protection scope of the present application.
Claims
1. A drug screening method based on liver cancer organoids, characterized in that: The steps include: S1, preparing liver cancer organoids from liver cancer tissues of different origins, wherein the liver cancer organoids prepared from liver cancer tissues of the same origin are recorded as a group; S2, obtaining pathological characteristics and genetic characteristics from the liver cancer organoid obtained in S1, analyzing the pathological characteristics and the genetic characteristics, and determining whether the liver cancer organoid can correspond to the pathological characteristics and the genetic characteristics of the liver cancer tissue of the same origin; S3, culturing the liver cancer organoids analyzed by the pathological characteristics and genetic characteristics in S2, then adding candidate drugs for treatment, and detecting cell viability and shape-based characteristic parameters of the liver cancer organoids; S4, calculating the IC50 value according to the cell viability test result, each liver cancer tissue of the same origin corresponds to an IC50 value and a characteristic parameter of the organoid based on morphology, and obtaining a candidate drug database based on liver cancer organoids by using the IC50 values corresponding to the liver cancer tissues of different origins and the characteristic parameters of the organoids based on morphology; S5, determining candidate drugs for treatment using the liver cancer organoid-based candidate drug database.
2. The drug screening method based on liver cancer organoids according to claim 1, characterized in that: The pathological characteristics include that the cell nuclei of the liver cancer organoids are large and have dense chromatin after HE staining, and the cells in the liver cancer organoids are irregular in shape or arranged in glandular tubules. The genetic characteristics include at least one of OPN, AFP, CEACAM6, and α-SMA.
3. The drug screening method based on liver cancer organoids according to claim 1, characterized in that: The morphologically based characteristic parameters of the organoid include at least one of a pseudoglandular whorl structure or a glandular cavity structure.
4. The drug screening method based on liver cancer organoids according to claim 3, characterized in that: The overall structure of the liver cancer organoids dissociates and collapses, the pseudo-glandular whorl structure or the glandular cavity structure disappears, and the number of living cells in the liver cancer organoids decreases.
5. The drug screening method based on liver cancer organoids according to any one of claims 1 to 4, characterized in that: S1 includes the following steps: S11, liver cancer tissues from different sources were dissociated and digested into single cells, and mixed with matrix gel to obtain a cell-matrix gel mixture; S12, solidifying the cell-matrix glue mixture obtained in S11 to obtain cell microspheres; S13, inoculating the cell microspheres obtained in S12 into a liver cancer organoid culture medium for culture, thereby obtaining liver cancer organoids.
6. The drug screening method based on liver cancer organoids according to claim 5, characterized in that: The liver cancer tissue described in S11 is human liver cancer tissue.
7. The drug screening method based on liver cancer organoids according to claim 5, characterized in that: During the mixing process of S11, every 5×10 4 -10×10 4 The single cells were mixed with 1 μL of the matrix gel.
8. The drug screening method based on liver cancer organoids according to claim 5, characterized in that: The flow rate of the cell-matrix gel mixture is 10-15 μL / min, and the flow rate of the fluorinated oil is controlled to be 100-110 μL / min.
9. The drug screening method based on liver cancer organoids according to claim 5, characterized in that: The particle size of the liver cancer organoid obtained in S14 is 450-550 μm.
10. The drug screening method based on liver cancer organoids according to claim 5, characterized in that: The cell microspheres described in S14 are inoculated into liver cancer organoid culture medium and cultured in an environment of 37-38.5° C. and 5%-7.5% CO 2 for 7-15 days.