Drug screening method based on colon cancer organs
Through the drug screening method based on colon cancer organoids, the problem that traditional models cannot accurately simulate the tumor microenvironment in vivo is solved, and the accuracy of drug screening and the provision of personalized treatments are achieved.
Patent Information
- Application Number
- CN202510361429.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-25
- Publication Date
- 2025-06-24
AI Technical Summary
In the prior art, traditional models of colon cancer research cannot accurately simulate the microenvironment of tumors in vivo, resulting in unpredictable treatment response and affecting the treatment effect.
Using a drug screening method based on colon cancer organoids, organoids prepared by colon cancer tissues of different sources are obtained, pathological, histological and genetic characteristics are obtained, drug screening is carried out, and IC50 values are calculated, candidate drug database is established, and suitable therapeutic drugs are determined.
It improves the accuracy and credibility of drug screening, and can determine the most suitable drugs based on the patient's own conditions and cancer development stage, and provides personalized treatment plans.
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Figure CN120193044A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the technical field of tumor organoids, and in particular to a drug screening method based on colon cancer organoids. Background Art
[0002] Colon cancer is one of the malignant tumors with high incidence and high mortality globally. Due to the differences among cancer patients, the treatment response of colon cancer patients is unpredictable, seriously affecting the treatment effect of colon cancer patients.
[0003] How to determine an ideal tumor model that accurately simulates the in vivo tumor microenvironment is crucial for providing personalized treatment for patients. In the prior art, the traditional models for colon cancer research are mainly two-dimensional tumor cell lines and xenograft tumor models. Two-dimensional tumor cell lines can neither simulate the spatial structure and heterogeneity of in vivo tumors nor reflect the interactions between tumor cells and between tumor cells and the tumor microenvironment. Although xenograft tumor models can highly simulate the structure, heterogeneity, and physiological environment of tumors, the immunodeficient mice used lack normal immune functions, making it impossible to conduct tumor immunity-related research and the development of immune drugs. At the same time, there are problems such as low modeling success rate, long experimental period, and high cost.
[0004] Therefore, it is urgent to select a suitable colon cancer research model to provide a new guiding direction for the personalized treatment of colon cancer. Summary of the Invention
[0005] To solve the problems in the prior art, this application provides a drug screening method based on colon cancer organoids.
[0006] This application provides a drug screening method based on colon cancer organoids, adopting the following technical solution:
[0007] A drug screening method based on colon cancer organoids includes the following steps:
[0008] S1, preparing colon cancer organoids from colon cancer tissues of different sources, where the colon cancer organoids prepared from colon cancer tissues of the same source are recorded as a group;
[0009] S2, obtaining pathological features, histological features, and genetic features from the colon cancer organoids obtained in S1, analyzing the pathological features, histological features, and genetic features, and determining that the colon cancer organoids can correspond to the pathological features, histological features, and genetic features of the colon cancer tissues of the same source;
[0010] S3, culturing the colon cancer organoids analyzed by the pathological features, histological features, and genetic features in S2, then adding candidate drugs for treatment, and detecting cell viability;
[0011] S4. Calculate the IC50 value according to the cell viability detection results. Each colon cancer tissue of the same origin corresponds to an IC50 value, and a candidate drug database based on colon cancer organoids is obtained by using the IC50 values corresponding to the colon cancer tissues of different origins.
[0012] S5. Determine the candidate drugs for treatment by using the candidate drug database based on colon cancer organoids.
[0013] 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.
[0014] The drug screening method based on colon cancer organoids provided by the present application uses the colon cancer organoids prepared from colon cancer tissues for anti-cancer drug screening, tests the cell viability after the action of candidate drugs, uses the IC50 value as the basis for determining the sensitivity of candidate drugs for colon cancer organoids from different patients, constructs a database covering the sensitive responses of candidate drugs for colon cancer organoids from different patients, each colon cancer tissue of a certain origin has a corresponding IC50 value, and each colon cancer tissue of a certain origin corresponds to a patient's own conditions and the development stage of colon cancer. Subsequently, according to the patient's own conditions and the corresponding stage of colon cancer development, the corresponding candidate drug with the smallest IC50 value is determined from the above database, and this candidate drug is used to treat patients with the same own conditions and the same cancer development stage, which can be a favorable tool for drug screening and personalized treatment of colon cancer patients.
[0015] Preferably, the pathological features include that the cell nuclei of the colon cancer organoids show atypia and different sizes, and the cell nuclei of the colon cancer organoids show deep nuclear staining and obvious nucleoli after HE staining;
[0016] The histological features include a three-dimensional structure similar to that of the colon cancer tissue, with a cavity-like or cyst-like morphology; the genetic features include at least one of FABP1 (intestinal epithelial cell marker), Lysozyme (Paneth cell marker), MUC1, CEACAM6 (colon cancer cell marker), CD3 (T cell marker), and CD68 (macrophage marker).
[0017] By comparing the above pathological features, histological features and genetic features, the present application confirms that the prepared colon cancer organoids can replace colon cancer tissues for drug screening, improving the accuracy and credibility of drug screening.
[0018] Preferably, S1 includes the following steps:
[0019] S11, Dissociate and digest colon cancer tissues from different sources into single cells;
[0020] S12, Mix the single cells obtained in S11 with Matrigel to obtain a cell-Matrigel mixture;
[0021] S13, Use microfluidic technology to shear the cell-Matrigel mixture obtained in S12 into droplets, and perform solidification treatment on the droplets to obtain cell microspheres;
[0022] S14, Inoculate the cell microspheres obtained in S13 into a colon cancer organoid culture medium for culture to obtain colon cancer organoids.
[0023] This application uses microfluidic technology to prepare colon cancer organoids from colon cancer tissues of different sources, which can achieve high-efficiency and high-throughput preparation of colon cancer organoids; under the same number of cells and time, the speed of constructing organoids using the method of this application can be increased by 2-3 times, and the prepared colon cancer organoids are uniform in size, the culture and maturation time is shortened. 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 colon cancer tissues according to the patient's own conditions, cancer development stage, etc., so as to construct a database with a wide range of data, providing a basis for the screening of candidate drugs for patients with different own conditions and different colon cancer stages in the future.
[0024] Preferably, the colon cancer tissue in S11 is human colon cancer tissue.
[0025] By using human colon cancer tissue to prepare colon cancer organoids, the formed colon cancer organoids retain the morphology and characteristics of the parent tissue, express colon-related cells and colon cancer cell markers, improve the accuracy of later drug sensitivity tests, improve the accuracy of the data in the database, and provide guarantee for the later drug screening of patients based on this database.
[0026] Preferably, during the mixing process of S12, every (5-10)×10 4 single cells are mixed with 1 μL of the Matrigel.
[0027] In the method for preparing colon cancer organoids of this application, colon cancer organoids with uniform size can still be obtained while using less Matrigel.
[0028] Preferably, the microfluidic technology used in S13 is a microfluidic technology based on the T-channel method.
[0029] Preferably, the flow rate of the cell-Matrigel mixture is 10-15 μL / min, and the flow rate of the oil-phase reagent is controlled to be 100-110 μL / min.
[0030] Preferably, the particle size of the colon cancer organoids obtained in S14 is 400-500 μm.
[0031] The morphology and structure of the colon 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 colon cancer organoids and enhancing the credibility of the drug screening database based on colon cancer organoids.
[0032] Preferably, after the cell microspheres in S14 are inoculated into the colon cancer organoid culture medium, they are cultured for 7-15 days in an environment of 35.5°C - 37.5°C and 4% - 5% CO2.
[0033] The culture time of the colon cancer organoids in this application is shortened. Compared with the traditional manual preparation of organoids, the organoid culture time is shortened from 30-60 days to 15 days. Description of the Drawings
[0034] Figure 1 It is the observation effect diagram of human-derived colon cancer organoids corresponding to 12 colon cancer patients under the microscope.
[0035] Figure 2 It is the HE staining effect diagram of human-derived colon cancer parent tissue (Figure a) and the corresponding human-derived colon cancer organoids (Figure b).
[0036] Figure 3 It is the IF detection of intestinal epithelial cells, colon cancer cells, and immune cell markers in human-derived colon cancer parent tissue and its organoids.
[0037] Figure 4 It is the candidate drug concentration-effect curve of human-derived colon cancer organoids after being treated with candidate drugs (the colon cancer organoids used in Figures a1, a2, and a3 correspond to the T1N0M1 stage, T2N1aM1 stage, and T3N1bM1 stage of the colon cancer tissue, and the candidate drug used is cisplatin; the colon cancer organoids used in Figures b1, b2, and b3 correspond to the T1N0M1 stage, T2N1aM1 stage, and T3N1bM1 stage of the colon cancer tissue, and the candidate drug used is tamoxifen; the colon cancer organoids used in Figures c1, c2, and c3 correspond to the T1N0M1 stage, T2N1aM1 stage, and T3N1bM1 stage of the colon cancer tissue, and the candidate drug used is anti PD-1). Detailed Embodiments
[0038] For better understanding and implementation, the technical solutions of this application will be clearly and completely described below in combination with embodiments. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all of the embodiments.
[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The terms used in the specification of this application are for the purpose of describing specific embodiments only and are not intended to limit this application.
[0040] Unless otherwise indicated, all numerical values expressing quantities of ingredients, reaction conditions, etc. used in the specification and claims are understood to be modified by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that can vary depending upon the desired properties sought to be obtained.
[0041] As used herein, "and / or" refers to one or all of the recited elements.
[0042] As used herein, "comprises" and "comprising" cover cases where only the recited elements are present and cases where there are also other unrecited elements in addition to the recited elements.
[0043] All percentages in this application are weight percentages, unless otherwise specified.
[0044] Unless otherwise indicated, the articles "a", "an", "the" and "said" as 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.
[0045] Example 1
[0046] This example presents a drug screening method based on colon cancer organoids:
[0047] The first step: construction of colon cancer organoids
[0048] The preparation method of colon cancer organoids comprises the following steps:
[0049] S11, single cell preparation: Twelve patients were numbered as P1, P2, P3, P4, P5, P6, P7, P8, P9, P10, P11, P12, and the specific information of the twelve patients is shown in Table 1;
[0050] Table 1
[0051]
[0052] Twelve human colon cancer tissues were separately taken and placed in 6-cm culture dishes. The tissue preservation solution was aspirated, and 3 mL of PBS was added to resuspend the tissue. The fat, blood stains and other debris on the tissue surface were removed with sterilized surgical scissors and forceps. Then the tissue was washed 3 times with 1 - 3 mL of PBS until the washing solution was clear, and the washing solution was removed. The tissue was resuspended with 1 - 3 mL of PBS containing 1% (v / v) PSA. 5 mL of PBS containing 1% (v / v) PSA was added to each well of a six-well cell culture plate. The tissue was clamped with forceps and slowly rotated clockwise 5 times in the well, and the same operation was repeated for each well. A new 6-cm culture dish was taken, and the washed tissue was transferred to the culture dish. 3 mL of PBS containing 1% (v / v) PSA was added to resuspend the tissue, which was cut into 2-mm fragments. The tissue and suspension were transferred to a 15-mL centrifuge tube with a pipette or Pasteur pipette, and the PBS was removed. The digestion solution was prepared: 10 mg of collagenase, 10 μL of DNase, and 10 μL of dispase were dissolved in 10 mL of DMEM / F12 medium containing 1% (v / v) BSA, used immediately, and filtered through a 22-μm filter membrane; 5 mL of the digestion solution was added to the centrifuge tube containing the tissue fragments, the tube cap was sealed with a sealing film, and it was placed in a shaker at 37°C, tilted, and incubated at 200 rpm for 15 - 30 min until it met the dissociation standard. 2.5 mL (half of the total volume of the digestion solution) of medium containing 10% serum was added to terminate the digestion. The obtained tissue suspension was centrifuged at 300 g for 10 min at 4°C, the supernatant was discarded, and the precipitate was resuspended with 5 mL of PBS containing 1% (v / v) PSA. A 100-μm cell strainer was placed on a 50-mL centrifuge tube with forceps, and the strainer was rinsed with 2 mL of PBS containing 1% (v / v) PSA. The resuspended tissue fluid was transferred to the cell strainer for filtration with a Pasteur pipette. The outer wall of the test tube was gently tapped to make the filtrate flow down slowly. The inner wall of the centrifuge tube used for digestion and the filtrate on the strainer were washed twice with an appropriate amount of PBS containing 1% (v / v) PSA. The collected tissue filtrate was centrifuged at 300 g for 10 min at 4°C, the supernatant was discarded, the number of red blood cells was observed, 1 - 3 mL of red blood cell lysate was added, and it was allowed to stand at room temperature for 3 min for lysis. An equal volume of PBS containing 1% (v / v) PSA was added to terminate the red blood cell lysis reaction, and it was centrifuged at 300 g for 5 min at 4°C in a centrifuge, and the supernatant was discarded. The cell precipitate was resuspended with 1 mL of complete culture medium. 10 μL of the cell suspension was added to 10 μL of AOPI (acid orange 7), and after thorough mixing, 20 μL of the mixture was injected onto a clean counting plate for cell counting.
[0053] S12, Preparation of cell-matrix gel mixture
[0054] Pre-cool 100 μL sterilized pipette tips at -20 °C in the refrigerator 1 day in advance, and pre-cool a 1 mL syringe containing 0.5 mL fluorinated oil on ice or in the -20 °C refrigerator 30 min in advance. Draw 5 mL of fluorinated oil with a 10 mL syringe, connect a needle and about 20 cm of polytetrafluoroethylene (PTFE) tubing, install it in the right hole of the three-way joint, install about 10 m of PTFE tubing in the left hole of the three-way joint, and install it in an injection pump outside the refrigerator (flow rate: 110 μL / min). Adjust the injection pump to fill the 20 cm PTFE tubing connected to the needle of the 10 mL syringe with fluorinated oil. Transfer the cell suspension into a 1.5 mL centrifuge tube, centrifuge at 300 g at room temperature for 3 min, discard the supernatant. According to the cell counting results, calculate the required volume of Matrigel at a concentration of 5×10 4 cells / μL, and add Matrigel to the cell pellet with a pre-cooled pipette tip. Gently stir several times on ice and then pipette to ensure no air bubbles are generated. Mix until there are no cell clumps to obtain a cell-Matrigel mixture.
[0055] S13, Preparation of cell microspheres
[0056] Inject the cell-Matrigel mixture into a pre-cooled 1 mL syringe containing fluorinated oil, connect a needle and about 10 cm of PTFE tubing, and then install it in an injection pump in the refrigerator (flow rate: 15 μL / min), install it in the middle hole of the three-way joint, and adjust the injection pump to make the cell-Matrigel mixture appear in this 10 cm PTFE tubing. Start the injection pump outside the refrigerator first, and then start the injection pump in the refrigerator. Shear the cell-Matrigel mixture into monodisperse droplets through the three-way tube, and then conduct them into about 10 m of PTFE tubing. When the PTFE tubing connected to the 1 mL syringe does not contain the cell-Matrigel mixture, stop both injection pumps. Place the PTFE tubing carrying the dispersed droplets of the cell-Matrigel mixture in a 37 °C incubator for 30 min to obtain cell microspheres.
[0057] S14, Preparation of human colon cancer organoids
[0058] Blow the cell microspheres into a petri dish containing human colon cancer organoid medium with a 10 mL syringe, and gently suck out the fluorinated oil on the surface of the petri dish with a 1 mL syringe with a needle. Place it in a 37 °C, 5.0% CO2 incubator for culture for 7 - 15 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.
[0059] After 7 days of culture, the colon cancer organoid model matures, the organoids are of uniform size, obvious structures appear inside the spheres, and the organoids from different patient sources have different morphologies (such as Figure 1 ), among which, most patients' organoids have a vesicular structure, and some patients' organoids (such as P3 and P4 in Figure 1 ) are of a dense structure.
[0060] Among them, the culture medium used in S14 is prepared according to the following formula: Add 100 μg / mL Primocin, 1% (v / v) GlutaMax, 1% (v / v) HEPES, 10 μM Y27632, 0.2 nM Wnt3a, 250 ng / mL R-Spondin-1, 100 ng / mL Noggin, 20 ng / mL FGF10, 5 ng / mL EGF, 1X B27 (containing vitamin A), 10 mM Nicotinamide, 1.25 mM N-acetylcysteine, 0.5 μg / mL Hydrocortisone, 10 μM Forskolin, 0.5 μM A8301, and 0.5 μM SB202190 to DMEM / F12 medium.
[0061] Analysis 1: HE staining of colon cancer organoids
[0062] Perform HE staining on the human colon cancer organoids prepared above, specifically including:
[0063] Step (1) Paraffin embedding of organoids
[0064] Preheat the embedding machine to 65 °C in advance.
[0065] Fixation: Add 4% PFA (about 200 μL), which is 10 times the volume of the organoids, to the EP tube and fix at room temperature for 20 min.
[0066] Preliminary 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.
[0067] Dehydration of organoids: Add 10 times the volume of 75% ethanol and dehydrate at room temperature for 5 min; aspirate the 75% ethanol, add 10 times the volume of 90% ethanol, and dehydrate at room temperature for 5 min.
[0068] Restaining of organoids: Add 20 μL of eosin solution and stain at room temperature for 5 min. (Restaining can be performed if the decolorization is severe).
[0069] Dehydration of organoids: Add 10 times the volume of absolute ethanol and dehydrate at room temperature for 5 min, repeating three times.
[0070] Clearing of organoids: Add 10 times the volume of xylene and soak at room temperature for 5 min, repeating three times.
[0071] Organoid paraffin embedding: 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, let it stand at 65 °C for 10 min, and after the paraffin solidifies, store the wax block at room temperature.
[0072] Step (2) Sectioning and HE staining
[0073] The paraffin-embedded block is sectioned on a microtome with a thickness of 5 μm.
[0074] Section dewaxing to water: 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 in sequence, and wash with distilled water.
[0075] 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 differentiating solution for 20 s, wash with tap water, blue with 1% ammonia water for 20 s, and wash with tap water.
[0076] Eosin staining of cytoplasm: Soak the sections in eosin staining solution and stain for 30 s.
[0077] Dehydration and mounting: Place the sections in 95% alcohol I for 5 min, 95% alcohol II for 5 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, xylene I for 5 min, xylene II for 5 min in sequence for dehydration and clearing. Take the sections out of xylene, place a clean blotting paper on the table, take out the glass slide from xylene and place it on the paper (with the section side up), quickly drop a drop of mounting medium in the center of the section, hold the forceps and gently clamp the right side of the cover glass, tilt it slightly so that its left side touches the mounting medium, and then slowly lower the cover glass to reduce or avoid generating air bubbles. Then perform microscopic examination and image acquisition and analysis.
[0078] The results of HE staining showed that the human colon cancer organoids highly reproduced the histological characteristics of the parental tumor, showing a vesicular structure, with the cell nuclei showing atypia, different sizes, deep nuclear staining, and obvious nucleoli (such as Figure 2 ).
[0079] Analysis 2: IF detection of human colon cancer organoids
[0080] Perform IF detection on the human colon cancer organoids prepared above, specifically including:
[0081] Step (1): Paraffin embedding of organoids
[0082] The specific steps are the same as step 1) in Characterization 1.
[0083] Step (2): Sectioning and IF detection
[0084] The paraffin sections are placed in an oven at 65 °C for 30 min - 1 h until the wax dissolves.
[0085] Deparaffinization of paraffin sections to water: The sections are successively placed in xylene I for 20 min, xylene II for 10 min, xylene III for 10 min, absolute ethanol for 10 min, 95% ethanol for 10 min, 70% ethanol for 10 min, and washed with distilled water for 5 min.
[0086] Antigen retrieval: The sections are placed in a retrieval box filled with EDTA antigen retrieval buffer (pH 8.0) and antigen retrieval is carried out in a microwave oven. High fire for 5 min, medium fire for 20 min, then low fire for 2 min. During this process, over-evaporation of the buffer should be prevented and the sections must not be dried. After natural cooling, the slides are placed in PBS (pH 7.4) and shaken on a shaker for 2 washes, 5 min each time.
[0087] Blocking: After taking out the sections from PBS and shaking off the liquid, use an immunohistochemistry pen to draw a circle around the tissue and add 100 μL of blocking solution (the blocking solution is PBS containing 5% BSA and 0.3% Triton X-100) to block for 1 hour.
[0088] Primary antibody incubation: Dilute the primary antibody to an appropriate concentration with the blocking solution, add 50 μL of the primary antibody to each section, place the sections flat in a humid box, and incubate overnight at 4 °C.
[0089] 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 section, and incubate at room temperature for half an hour.
[0090] Counterstaining with DAPI: Wash 3 times with PBST, 10 min each time, add the diluted DAPI, and incubate at room temperature for 10 min. Wash with PBS for 5 min.
[0091] Mounting and microscopic examination: Add about 30 μL of anti-fluorescence quenching agent to each section, cover with a coverslip, observe under a fluorescence microscope, and take pictures for recording.
[0092] IF detects the expression of intestinal epithelial cell markers and colon cancer markers in the parental tissue of colon cancer and its organoids. The results show that both the parental tissue and the corresponding colon cancer organoids express FABP1 (intestinal epithelial cell marker), Lysozyme (Paneth cell marker); highly express MUC1 and CEACAM6 (colon cancer cell markers), and both express CD3 (T cell marker) and CD68 (macrophage marker); all of which indicate that the organoids reproduce the genetic characteristics of cancer cells in the tumor tissue (such as Figure 3 ).
[0093] Step 2: Drug screening method based on colon cancer organoids
[0094] Place the human colon cancer organoids with growing structures in 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 equilibrate the temperature of the CTG substrate to room temperature; mix the CTG buffer and the CTG substrate in a light-protected bottle and gently vortex to form the CTG reagent; equilibrate the temperature of the opaque 96-well plate at room temperature for 30 min; add 100 μL of the 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 a microplate reader, make an IC 50 curve, read the IC50 values corresponding to different candidate drugs, and obtain a candidate drug database based on different colon cancer organoids (such as Figure 4 ).
[0095] Therefore, for colon cancer tissues at the T1N0M1 stage, the candidate drug should be anti-PD-1 with the smallest IC50 value; for colon cancer tissues at the T2N1aM1 stage, the candidate drug should be cisplatin with the smallest IC50 value; for colon cancer tissues at the T3N1bM1 stage, the candidate drug should be cisplatin with the smallest IC50 value.
[0096] Subsequently, according to the individual conditions and cancer development stages of different patients, determine the candidate drugs suitable for the patients from the above database based on the IC50 values (select the candidate drug with the smallest IC50 value), and use this candidate drug to treat the corresponding colon cancer patients.
[0097] 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 colon cancer organoids, characterized in that: The steps include: S1, preparing colon cancer organoids from colon cancer tissues of different sources, wherein the colon cancer organoids prepared from colon cancer tissues of the same source are recorded as a group; S2, obtaining pathological characteristics, histological characteristics and genetic characteristics from the colon cancer organoid obtained in S1, analyzing the pathological characteristics, the histological characteristics and the genetic characteristics, and determining whether the colon cancer organoid can correspond to the pathological characteristics, the histological characteristics and the genetic characteristics of the colon cancer tissue of the same source; S3, culturing the colon cancer organoids analyzed by the pathological characteristics, histological characteristics and genetic characteristics in S2, then adding candidate drugs for treatment, and detecting cell viability; S4, calculating the IC50 value according to the cell viability test results, each colon cancer tissue of the same origin corresponds to an IC50 value, and using the IC50 values corresponding to the colon cancer tissues of different origins to obtain a candidate drug database based on colon cancer organoids; S5, determining candidate drugs for treatment using the colon cancer organoid-based candidate drug database.
2. The drug screening method based on colon cancer organoids according to claim 1, characterized in that: The pathological characteristics include that the cell nuclei of the colon cancer organoids are heterogeneous and of different sizes, and the cell nuclei of the colon cancer organoids are darkly stained and have obvious nucleoli after HE staining; The histological features include a three-dimensional structure similar to the colon cancer tissue, having a luminal or cystic morphology; The genetic characteristics include at least one of FABP1, Lysozyme, MUC1, CEACAM6, CD3, and CD68.
3. The drug screening method based on colon cancer organoids according to any one of claims 1-2, characterized in that: S1 includes the following steps: S11, colon cancer tissues from different sources were dissociated and digested into single cells; S12, mixing the single cell obtained in S11 with matrix gel to obtain a cell-matrix gel mixture; S13, shearing the cell-matrix gel mixture obtained in S12 into droplets using microfluidic technology, and solidifying the droplets to obtain cell microspheres; S14, inoculating the cell microspheres obtained in S13 into a colon cancer organoid culture medium for culture, thereby obtaining colon cancer organoids.
4. The drug screening method based on colon cancer organoids according to claim 3, characterized in that: The colon cancer tissue described in S11 is human colon cancer tissue.
5. The drug screening method based on colon cancer organoids according to claim 3, characterized in that: During the mixing process of S12, every (5-10)×10 4 The single cells were mixed with 1 μL of the matrix gel.
6. The drug screening method based on colon cancer organoids according to claim 3, characterized in that: The microfluidic technology used in S13 is a microfluidic technology based on the T-channel method.
7. The drug screening method based on colon cancer organoids according to claim 6, characterized in that: The flow rate of the cell-matrix gel mixture is 10-15 μL / min, and the flow rate of the oil phase reagent is controlled to be 100-110 μL / min.
8. The drug screening method based on colon cancer organoids according to claim 3, characterized in that: The particle size of the colon cancer organoid obtained in S14 is 400-500 μm.
9. The drug screening method based on colon cancer organoids according to claim 3, characterized in that: The cell microspheres described in S14 are inoculated into a colon cancer organoid culture medium and cultured in an environment of 35.5° C.-37.5° C. and 4%-5% CO 2 for 7-15 days.