Method for constructing tumor organoid by using kindlin-2 high expression system

Abstract

The invention discloses a method for constructing a tumor organoid by using a kindlin-2 high expression system. The method comprises the following steps: S1, cutting an obtained tumor tissue into pieces; adding collagenase for hydrolysis, and sieving to obtain a cell suspension; s2, centrifuging the cell suspension, absorbing supernate, retaining tumor cell precipitates, transfecting tumor cells by using a kindlin-2 virus, and performing high expression of kindlin-2 protein; s3, uniformly mixing the tumor cells with high expression of kindlin-2 with matrigel according to a set proportion to obtain a mixed solution, and putting the mixed solution on ice; s4, transferring the mixed solution into a cell culture plate, adding a culture medium for culturing after solidification, and culturing for a period of time to obtain the tumor organoid. According to the invention, the kindlin-2 protein is highly expressed in tumor cells, stromal stem cells or tumor-related fibroblasts so as to promote the construction of a model of tumor organs, especially for the organs which are difficult to construct by conventional methods, the construction of the tumor organs by using the kindlin-2 high expression system represents a valuable early clinical tool, and the construction of the tumor organs is very convenient. The method is used for researching a tumor evolution mechanism.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to a method for constructing tumor organoids using a kindlin-2 high expression system. Background Art

[0002] Tumor organoids represent three-dimensional cultures of tumor cells propagated directly from tissue samples from lung cancer patients. These organoids are grown under precise conditions that preserve tissue architecture, genetic variation, and sensitivity to drugs similar to those found in the original tumor. Serving as a system for rapid screening of prospective therapeutic approaches, these patient-derived models provide scientists with a means to test the efficacy and safety of new drugs in models that closely resemble actual tumors found in the body. Additionally, tumor organoids contain a variety of cell populations found in tumor tissue, which is critical for studying the basic mechanisms of disease evolution and the emergence of drug resistance. By analyzing the genetic and molecular features of patient-derived tumor organoids, researchers can identify biomarkers that predict treatment response or prognosis. Summary of the invention

[0003] The present invention discloses a method for constructing tumor organoids using a kindlin-2 high expression system, and a method for constructing patient-derived tumor organoids. The present invention highly expresses kindlin-2 protein in patient-derived tumor cells, stromal stem cells, or tumor-associated fibroblasts, thereby promoting the construction model of tumor organs, especially for those organoids that are difficult to construct using conventional methods. The construction of tumor organoids using a kindlin-2 high expression system will represent a valuable preclinical tool for studying the mechanism of tumor evolution and promoting the development of precision medicine.

[0004] To achieve the above object, the present invention proposes a method for constructing tumor organoids using a kindlin-2 high expression system, comprising the following steps: S1: mince the obtained tumor tissue, add collagenase to hydrolyze it, and sieve to obtain a cell suspension; S2: centrifuging the cell suspension, removing the supernatant and retaining the tumor cell precipitate, transfecting one of the tumor cells, stromal stem cells and tumor-associated fibroblasts with the kindlin-2 virus to obtain a tumor cell precipitate with high expression of kindlin-2; S3: Mix the tumor cell precipitate with high expression of kindlin-2 and the matrix gel according to the set ratio to obtain a mixed solution, and place the mixed solution on ice; S4: The mixed solution is transferred to a cell culture plate, and after solidification, culture medium is added for culture. After a period of culture, tumor organoids are obtained.

[0005] Preferably, in step S1, the source of the obtained tumor tissue is cancerous tissue, including one of lung cancer, esophageal cancer, thyroid cancer, pancreatic cancer, tongue cancer, gastric cancer, liver cancer, intestinal cancer, breast cancer, sarcoma, nasopharyngeal cancer, ovarian cancer and cervical cancer tissue.

[0006] Preferably, the kindlin-2 high expression system comprises one of lentivirus, adenovirus, adeno-associated virus, and retrovirus vector tools.

[0007] Preferably, in step S1, the tissue is cut into pieces of 0.5-2 mm. 3 Add 10 ml collagenase to the small fragments or paste, place it in a 37°C cell culture incubator for digestion for 30 minutes; then add complete culture medium to terminate the digestion, and use a 70-100 mesh cell sieve to filter the digested tissue to obtain a cell suspension.

[0008] Preferably, in step S2, when kindlin-2 is highly expressed in stromal stem cells or tumor-associated fibroblasts, the stromal stem cells or tumor-associated fibroblasts highly expressing kindlin-2 need to be mixed with tumor cells in a certain ratio, and this ratio is generally 30%-60% of the stromal stem cells or tumor-associated fibroblasts highly expressing kindlin-2 to the total number of cells.

[0009] Preferably, in step S3, the tumor cell pellet is washed with PBS, and then the amount of organoid matrix gel to be added is calculated according to the ratio of 8,000 to 10,000 cells per well to 10-30 µl of matrix gel.

[0010] Preferably, in step S4, a mixture of matrix gel and cells is aspirated with a pipette and transferred to a cell culture well plate, and the mixed suspension is dotted to the bottom of the culture well, and it cannot touch the side wall of the culture well after spreading; the culture plate is placed in a cell culture incubator for solidification for 30 minutes, and after the matrix gel solidifies and no longer flows, culture medium is slowly added along the well wall, and the culture medium is replaced every 3-4 days to finally obtain tumor organoids.

[0011] Preferably, the culture medium comprises the following reagents: the culture medium contains 10 µMol Y-27632-HCl, 10% fetal bovine serum; the culture medium is prepared and stored at 4°C for no more than 7 days.

[0012] Preferably, the matrix gel comprises Matrigel, Cultrex BME, collagen, gelatin and other synthetic matrix gels.

[0013] The benefits of the present application are as follows: the present application utilizes kindlin-2 to construct a high expression system, participates in integrin-mediated signal transduction, and serves as a bridge between the extracellular matrix and intracellular pathways; kindlin-2 plays a key role in the activation of lung fibroblasts and the progression of pulmonary fibrosis. Kindlin-2 is essential for the enhancement and development of focal adhesions and stress fibers of activated fibroblasts and myofibroblasts. The increase in kindlin-2 is also associated with the epithelial-mesenchymal transition and immunosuppression of gastric cancer. Kindlin-2 is widely and highly expressed in tumor cells of various cancer types such as lung cancer, pancreatic cancer, breast cancer, colon cancer, and liver cancer; it is also highly expressed in tumor stromal cells such as tumor-associated fibroblasts and stromal stem cells. The organoids constructed by the present invention can be used to analyze the biological characteristics of human tumor organoids; a platform for predicting clinical tumor drug sensitivity is also established using tumor organoids as a model tool, which more systematically and comprehensively evaluates the effectiveness of treatment plans, and provides support for screening the most suitable and optimal individualized precision treatment plans for tumor patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is a schematic diagram of the steps of the present invention; Figure 2-4 It is a test diagram of the implementation results of the present invention. DETAILED DESCRIPTION

[0015] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] The terms "include", "including", "have", "contain", etc. used herein are open-ended terms, which mean including but not limited to. For those skilled in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0017] In addition, if there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, if the meaning of "and / or" appearing in the full text is to include three parallel schemes, taking "A and / or B" as an example, it includes scheme A, or scheme B, or a scheme that satisfies both A and B. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0018] Tumor cells are taken from tissue samples of lung cancer patients and cultured in three dimensions. These organoids are grown under precise conditions that preserve tissue architecture, genetic variation, and sensitivity to drugs similar to those found in the original tumor. These patient-derived models serve as a system for rapid screening of prospective treatments, providing scientists with a means to test the efficacy and safety of new drugs in models that closely resemble actual tumors found in the body. Additionally, tumor organoids contain a variety of cell populations found in tumor tissue, which is critical for studying the basic mechanisms of disease evolution and the emergence of drug resistance. By analyzing the genetic and molecular features of patient-derived tumor organoids, researchers can identify biomarkers that predict treatment response or prognosis.

[0019] In order to implement the above solution, the technical solution adopted by this application includes the following steps: 1. Method for constructing lung cancer organoids by expressing kindlin-2 in lung cancer cells 1. Primary Tissue Collection and Processing First, mince the tumor tissue into pieces of approximately 0.5-2 mm. 3 Add 10 ml collagenase to the tissue culture incubator at 37°C and digest for about 30 minutes. Add 10 ml culture medium to stop digestion, filter the digested tissue with a 70-100 mesh cell sieve to obtain a cell suspension; centrifuge the cell suspension (1000 rpm, 10 min) and remove the supernatant, wash the cells twice with phosphate buffer, and try to remove the residual PBS buffer after the last centrifugation.

[0020] 2. Matrigel encapsulation of lung cancer cells with high expression of kindlin-2 Centrifuge the digested lung cancer cells at 200-300g for 3-5 minutes, remove the supernatant and retain the precipitate, add the corresponding amount of organoid matrix gel (taking 24-well plate as an example) according to the ratio of 8000-10000 cells per well to 10-30μL matrix gel, and mix on ice. Be gentle when mixing and blowing, and do not generate a large number of bubbles. If mixing at room temperature, it needs to be controlled within 15 seconds. After mixing, place on ice. It is particularly important to note that in order to ensure the structural stability of the organoid matrix gel during the culture process, the volume ratio of the matrix gel should be kept above 70%.

[0021] 3. Seed plate culture Use a pipette to absorb the mixture of matrix gel and cells and transfer it to the cell culture well plate (e.g. 10-30 μL of mixed suspension is applied to each well of a 24-well cell culture plate). The mixed suspension is applied to the bottom of the culture well. It should not touch the side wall of the culture well after spreading. Place the culture plate in a 37°C, 5% CO2 cell culture incubator to solidify for 30 minutes. After the matrix gel solidifies and no longer flows, slowly add complete culture medium along the well wall (for example, add 1 mL of complete culture medium for 24 wells), place the 24-well plate in a 5% CO2 cell culture incubator, and replace the complete culture medium every 3-4 days.

[0022] In the specific implementation process, the culture medium contains 10 µmol Y-27632-HCl and 10% fetal bovine serum. The culture medium is stored at low temperature after preparation and is stored at 4°C for no more than 7 days to avoid bacterial infection of the culture medium.

[0023] In actual use, tumor organoid culture includes but is not limited to lung cancer, esophageal cancer, thyroid cancer, pancreatic cancer, tongue cancer, intestinal cancer, breast cancer, cervical cancer, etc.; the kindlin-2 high expression system used can be a lentivirus, adenovirus packaging vector or other vector; when kindlin-2 is highly expressed in stromal stem cells or tumor-associated fibroblasts, it is necessary to mix the stromal stem cells or tumor-associated fibroblasts that highly express kindlin-2 with tumor cells in a certain proportion, which is generally 30%-60% of the total number of cells.

[0024] 2. Methods for establishing a drug sensitivity detection platform for human lung cancer organoids 1. Organoid drug sensitivity screening (96-well plate) (1) Calculate the required cell volume based on the number of drugs to be screened, concentration, and number of repetitions. Here, 10,000 single cells per well are used as a reference. Drug addition and organoid plating design: The designed concentration is 10 μM for doxorubicin, cisplatin, 5-fluorouracil, erlotinib, and paclitaxel (Table 1). Each drug is set with 5 replicates, and the experiment is repeated three times independently on a 96-well plate.

[0025] (2) Collecting organoids: Aspirate the original organoid culture medium, add 500 μL of pre-cooled basal culture medium DMEM / F12 to each well, and use a 1 mL pipette tip to blow and scrape the matrix gel to remove the matrix gel and organoids; transfer the matrix gel containing the organoids to a 15 mL centrifuge tube, wash the well plate with pre-cooled 1× PBS and combine the organoid suspension; insert the 15 mL centrifuge tube on ice to soften the matrix gel; use a 1 mL pipette tip to blow the organoid suspension 15 times to separate the organoids from the matrix gel; incubate at 4°C, 30 Centrifuge at 0g for 3 min, remove the supernatant and add 10mL of pre-cooled 1×PBS to resuspend the organoids; centrifuge at 4℃ and 300g for 3 min, remove the supernatant and add 3mL of organoid digestion solution, and place in a 37℃ incubator for digestion for 3-6min; blow the organoids until the organoids are digested into small cell clusters or single cells; add 6-8mL of basal culture medium DMEM / F12 and 0.2mL of fetal bovine serum to terminate digestion (microscope digestion effect); after mixing, centrifuge at 4℃ and 300g for 3 min, remove the supernatant and wash twice with basal culture medium DMEM / F12; resuspend the cells with 1mL of tumor organoid basal culture medium and transfer them to a 1.5mL EP tube, take 18μL of cell suspension (add 2μL of trypan blue) for live cell detection and counting.

[0026] (3) Organoid 96-well plate plating: Calculate the amount of matrix gel according to the number of wells required for drug screening, drop the organoid into the 96-well plate at 10 μL per well, and solidify in the incubator for 15 minutes; add 110 μL of organoid complete growth medium to each well and culture in the incubator for 7-10 days; dilute and add drugs, the concentrations of the five drug combinations are shown in the table below. After adding drugs, continue to culture the organoids in the incubator for 3-5 days.

[0027] Table 1: Concentration and function of added drugs

[0028] 2. Organoid Viability Assay Use the CellTiter-Lumi™ 3D Cell Viability Luminescence Kit to assess cell viability. Balance the temperature of the 3D Cell Viability Detection Reagent in a 20°C water bath or at room temperature; take out the lung cancer organoid culture plate and place it at room temperature for 5 minutes to allow the plate temperature to equilibrate with room temperature; turn the 3D Cell Viability Detection Reagent upside down to mix evenly; add 50μL of 3D Cell Viability Detection Reagent to each well to be tested in the 96-well plate; oscillate in an ELISA reader for 6 minutes, let it stand for 12 minutes, and then measure the chemiluminescence value; calculate the drug inhibition rate and IC50 corresponding to each drug concentration in the experimental group.

[0029] The calculation formula of survival rate is: Survival rate = [(experimental group - blank control) - (negative control - blank control)] / [(positive control - blank control) - (negative control - blank control)]; Through relevant experiments and verification, first, for tumor samples that had not formed lung cancer organoids, when kindlin-2 was highly expressed in tumor cells, tumor organoids were successfully obtained. Figure 2 The left picture shows that organoids failed to form after empty vector transfection; however, organoids were significantly formed after overexpression of kindlin-2 ( Figure 2 (middle picture). Figure 2 In the right figure, LCCs are lung cancer tumor cells; Flag is lung cancer cells transfected with a vector carrying Flag as a control, and Flag-K2 represents a vector expressing kindlin-2.

[0030] See also Figure 3 and Figure 4 The five drugs were used to treat the cell viability of the tumor organoids constructed by high expression of kindlin-2 in lung cancer cells. The results showed that in the lung cancer organoids constructed by high expression of kindlin-2 in lung cancer cells, 5-fluorouracil, doxorubicin, cisplatin, erlotinib and paclitaxel had different degrees of drug sensitivity. LCO-48,49 are lung cancer patient cells that cannot be formed by traditional organoid culture methods.

[0031] The above descriptions are only optional embodiments of the present invention, and are not intended to limit the patent scope of the present invention. All equivalent structural changes made using the contents of the present invention's specification and drawings, or directly / indirectly applied in other related technical fields, are included in the patent protection scope of the present invention.

Claims

1. A method for constructing tumor organoids using a kindlin-2 high expression system, characterized in that: The following steps are involved: S1: mince the obtained tumor tissue, add collagenase to hydrolyze it, and sieve to obtain a cell suspension; S2: centrifuging the cell suspension, removing the supernatant and retaining the tumor cell precipitate, transfecting one of the tumor cells, stromal stem cells and tumor-associated fibroblasts with the kindlin-2 virus to obtain a tumor cell precipitate with high expression of kindlin-2; S3: Mix the tumor cell precipitate with high expression of kindlin-2 and the matrix gel according to the set ratio to obtain a mixed solution, and place the mixed solution on ice; S4: The mixed solution is transferred to a cell culture plate, and after solidification, culture medium is added for culture. After a period of culture, tumor organoids are obtained.

2. The method according to claim 1, characterized in that In step S1, the source of the obtained tumor tissue is cancerous tissue, and the tumor includes one of lung cancer, esophageal cancer, thyroid cancer, pancreatic cancer, tongue cancer, gastric cancer, liver cancer, intestinal cancer, breast cancer, sarcoma, nasopharyngeal cancer, ovarian cancer and cervical cancer.

3. The method according to claim 1, characterized in that The kindlin-2 high expression system includes one of lentivirus, adenovirus, adeno-associated virus, and retrovirus vector tools.

4. The method according to claim 1, characterized in that: In step S1, the tissue was minced into pieces of 0.5-2 mm. 3 Add 10 ml collagenase to the small fragments or paste, place it in a 37°C cell culture incubator for digestion for 30 minutes; then add complete culture medium to terminate the digestion, and use a 70-100 mesh cell sieve to filter the digested tissue to obtain a cell suspension.

5. The method according to claim 1, characterized in that In step S2, when kindlin-2 is highly expressed in stromal stem cells or tumor-associated fibroblasts, it is necessary to mix the stromal stem cells or tumor-associated fibroblasts highly expressing kindlin-2 with tumor cells in a certain ratio, and this ratio is generally 30%-60% of the total number of cells in the stromal stem cells or tumor-associated fibroblasts highly expressing kindlin-2.

6. The method according to claim 1, characterized in that In step S3, the tumor cell pellet is washed with PBS, and then the amount of organoid matrix gel to be added is calculated according to the ratio of 8,000 to 10,000 cells per well to 10-30 ul of matrix gel.

7. The method according to claim 1, characterized in that: In step S4, use a pipette to suck up the mixture of matrix gel and cells and move it to the cell culture well plate, and dot the mixed suspension to the bottom of the culture well. After spreading, it cannot touch the side wall of the culture well; put the culture plate in a cell culture incubator to solidify for 30 minutes. After the matrix gel solidifies and no longer flows, slowly add culture medium along the wall of the well. Replace the culture medium every 3-4 days to finally obtain tumor organoids.

8. The method according to claim 1, characterized in that: The culture medium contains the following reagents: The culture medium contains 10 µM Y-27632-HCl, 10% fetal bovine serum; the culture medium is prepared and stored at 4°C for no more than 7 days.

9. The method according to claim 1, characterized in that: Matrix gels include Matrigel, Cultrex BME, collagen, gelatin and other synthetic matrix gels.

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