A standardized method for constructing lung cancer pleural effusion organoids with high clinical consistency and its application in drug susceptibility testing.

Abstract

This invention belongs to the field of biotechnology and discloses a standardized method for constructing lung cancer pleural effusion organoids with high clinical consistency and its application in drug sensitivity testing. The method includes the following steps: density gradient centrifugation of lung cancer pleural effusion samples using a dual-concentration Percoll layer, collecting the cell layer at the interface between the two Percoll layers; and centrifuging the collected cells at a concentration of 1-5 × 10⁻⁶ cells / mL. 5 The tissue was seeded at a density of 1 organoid / 50 μL of matrix gel and incubated to solidify. The tissue was then cultured in complete culture medium. Organoids from lung cancer pleural effusion were collected. The cultured organoids were identified and verified against the original lung cancer tissue using whole-exome sequencing. Drug sensitivity testing was performed on the organoids. This invention standardizes parameters for each step from sample pretreatment to drug sensitivity testing. Through optimized Percoll density gradient centrifugation, highly efficient enrichment of tumor cells and removal of impurities were achieved, resulting in extremely high initial effective tumor cell clusters and significantly faster organoid growth.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and precision medicine, and in particular, it relates to a standardized method for constructing lung cancer pleural effusion organoids with high clinical consistency and its application in drug sensitivity testing. Background Technology

[0002] In the context of precision medicine, the need for drug sensitivity prediction in personalized lung cancer treatment is becoming increasingly urgent. However, existing technologies face the following bottlenecks: 1. Difficulty in obtaining samples: Patients with advanced lung cancer often lose the opportunity for surgery, making it difficult to obtain tissue specimens; while pleural fluid samples are highly accessible, their cellular composition is complex (containing a large number of red blood cells, immune cells and stromal cells), resulting in low purity of tumor cells and unstable modeling success rate.

[0003] 2. Lack of model fidelity: Traditional 2D culture cannot simulate the three-dimensional microenvironment in vivo, resulting in the distortion of cell function; while existing 3D culture technology lacks unified standards, has large differences in operation, and the culture medium formulation is not targeted enough, making it difficult to ensure the genetic and phenotypic consistency between organoids and primary tumors.

[0004] 3. Limited identification system: Existing technologies rely heavily on morphological observation and lack an integrated evaluation from genomics to function, making it difficult to prove the true predictive value of in vitro drug sensitivity results for clinical efficacy.

[0005] In summary, existing technologies suffer from drawbacks such as low sample processing efficiency, unreasonable culture systems, imperfect identification systems, lack of drug sensitivity data, weak clinical relevance of existing models, and lack of standardized operating procedures in the process of constructing organoids from lung cancer pleural effusion. Therefore, there is an urgent need for one or more related new methods or reagents.

[0006] A search revealed no patent publications related to this invention's patent application. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a standardized method for constructing lung cancer pleural effusion organoids with high clinical consistency and its application in drug sensitivity testing. By optimizing the cell separation process and culture medium formulation, and combining the "pathology-gene-function" three-in-one identification strategy, a high-fidelity and reproducible in vitro lung cancer model can be constructed.

[0008] The technical solution adopted by this invention to solve its technical problem is: A standardized method for constructing lung cancer pleural effusion organoids with high clinical consistency includes the following steps: (1) Cell separation: The lung cancer pleural fluid sample was subjected to density gradient centrifugation. The centrifugation used a dual-concentration Percoll layer with a bottom layer of 75% mass concentration and a middle layer of 40% mass concentration. The mixture was centrifuged at 800g for 20min and the cell layer at the interface between the two Percoll layers was collected. (2) Inoculation: The collected cells are inoculated at a rate of 1-5 × 10⁻⁶. 5 Inoculate the culture plate at a density of 1 per 50 μL of matrix gel and incubate until solidification. (3) Culture: Cultured using a complete culture medium, which includes basal culture medium, B27, N2, nicotinamide, N-acetyl-L-cysteine, A83-01, Y27632, Noggin, EGF, Wnt-3a, R-spondin1, FGF7, FGF10 and lung cancer pleural effusion supernatant with a volume fraction of 5%-20%; (4) Establish an organoid model of pleural effusion in lung cancer; (5) Multidimensional identification: HE staining pathological identification, IHC marker identification and whole exon sequencing verification were performed on the cultured organoids. The verification was successful, and lung cancer pleural effusion organoids with high clinical consistency were obtained.

[0009] Furthermore, the specific steps of step (1) are as follows: ① Take human lung cancer pleural effusion samples, aliquot them into 50 mL centrifuge tubes, centrifuge at 500g for 5 min, discard the supernatant, and collect the cell pellet; ② Add an appropriate amount of tissue / organoid washing solution to the cell pellet for resuspending, filter using a 100 μm cell sieve, collect the filtrate in a centrifuge tube, centrifuge again at 500g for 5 min, discard the supernatant and add 5 mL of tissue / organoid washing solution to resuspend the cell pellet. ③ Resuspend the precipitate with 10 mL of 75% Percoll solution and transfer it to a new 50 mL centrifuge tube. Then slowly add 10 mL of 40% Percoll solution to the top layer to create a density gradient. ④ Slowly add a mixture of cells and tissue / organoid washing solution to the upper layer of a 40% Percoll solution. After centrifugation at 800g for 20 min, cells are separated into layers based on density differences. ⑤ Collect the top and middle layers rich in tumor cells, transfer them to a new 15 mL centrifuge tube, add 5 mL of tissue / organoid washing solution and mix well to obtain a cell suspension for later use.

[0010] Furthermore, the formulation of the tissue / organoid washing solution in step ② includes: 1×GlutaMAX, 1×HEPES, and 1×penicillin-streptomycin added to DMEM culture medium.

[0011] Furthermore, the specific steps of step (2) are as follows: ① Take 30 μL of cell suspension and mix it with an equal volume of organoid fluorescent dye, then use an organoid counter to count the cells; ② Each 50 μL Matrigel gel contains 1×10 5 Prepare a mixture in proportion of individual cells or cell clusters, and inoculate it into a 24-well culture plate at a rate of 50 μL per well. ③ Incubate the culture plate in a 37℃, 5% CO2 incubator for 15 minutes to allow the Matrigel to solidify completely, thereby creating a three-dimensional growth environment and promoting organoid formation; The specific steps of step (3) are as follows: Organoid culture: ① Add 500 μL of complete lung cancer organoid culture medium to each well of the culture plate in step (2) ③ above; ② Continue to incubate the culture plate in a 37℃, 5% CO2 incubator; ③ Replace the culture medium with fresh one every 2–3 days, and regularly observe and record the growth status of organoids.

[0012] Furthermore, the formulation of the complete lung cancer organoid culture medium is as follows: The organoid basal medium was Advanced DMEM / F12, supplemented with the following components: 1×Glutamax supplement, 1×HEPES, 1×Penicillin-streptomycin, 1×B27 additive, 1×N2 additive, and various factors in the following concentration ranges: 1–100 mM Nicotinamide, 1–10 mM N-acetyl-L-cysteine, 1–1000 nM TGF-β receptor inhibitor (A83-01, i.e., TGF-β type I receptor inhibitor A83-01), 1–50 μM ROCK kinase inhibitor (Y27632), 1–100 ng / mL Human Noggin, 1–100 ng / mL Human Epidermal Growth Factor (EGF), and 1–500 ng / mL... Wnt-3a protein, 1-500 ng / mL R-spondin1 protein, and pleural effusion supernatant at concentrations of 1-100% by volume.

[0013] Furthermore, the formulation of the complete lung cancer organoid culture medium is as follows: The basal culture medium consisted of Advanced DMEM / F12, 1× glutamine supplement, 1× hydroxyethylpiperazine ethanesulfonic acid, 1× penicillin-streptomycin, 1× B27 additive, 1× N2 additive, 10 mM nicotinamide, 1.25 mM N-acetyl-L-cysteine, 500 nM TGF-β receptor inhibitor, 10 μM ROCK kinase inhibitor, 25 ng / mL human recombinant Noggin protein, 25 ng / mL human epidermal growth factor, 100 ng / mL Wnt-3a protein, 250 ng / mL R-spondin1 protein, and pleural effusion supernatant at a final volume concentration of 10%.

[0014] Furthermore, the specific steps of step (4) are as follows: ① Use pre-cooled tissue / organoid washing solution to release the organoids from the matrix gel by repeated blowing; ② Centrifuge and discard the supernatant, then wash with PBS to remove residual matrix gel; Alternatively, the specific steps of step (5) are as follows: 1) Take 5 × 10 5 Each organoid was treated with 500 μL of tissue fixative (4% paraformaldehyde) for subsequent HE staining and IHC identification. 2) Take 5 × 10 5 For each organoid, add 500 μL of RNA stabilization solution such as RNAlater™ Stabilization (Thermo Fisher SCIENTIFIC product) for genome sequencing analysis.

[0015] The lung cancer pleural effusion organoids with high clinical consistency were constructed using the method described above.

[0016] The application of organoids from pleural effusion in lung cancer in personalized drug screening, as described above.

[0017] The method for screening personalized medicines for lung cancer using organoids derived from pleural effusion, as described above, includes the following steps: ① Select organoids, and use 500 organoids per well, 10% Matrigel gel and complete culture medium to make a 50 μL system, and seed it in a 384-well plate; ② Place the culture plate in a 37℃, 5% CO2 incubator and incubate overnight for 1 day; ③ Design drug treatment protocols: Design drug sensitivity protocols, with five concentration gradients for each drug as a single agent (0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM) and one control group (0 μM). For combination drugs, five concentration gradients are set up (0.0625 μM, 0.25 μM, 1 μM, 4 μM, 16 μM) and one control group (0 μM). Each condition is prepared in 5 replicates and added to 50 μL of culture system. Drug sensitivity testing is performed from 72h to 144h of culture. During this window period, organoids are in the logarithmic growth phase and are most sensitive to drugs, which can most realistically reflect the cell behavior under clinical chemotherapy stress. ④ After adding the drug, place it in a 37℃, 5% CO2 incubator and continue culturing for 6 days; ⑤ Add 40 μL of detection reagent to each well and incubate with a horizontal shaker for 10 minutes to ensure the reagent is fully reacted; ⑥ Use an enzyme-linked immunosorbent assay (ELISA) reader to detect organoid viability using chemiluminescence immunoassay; ⑦ Conduct data analysis to assess drug sensitivity.

[0018] The advantages and positive effects of this invention are as follows: 1. This invention establishes a highly standardized end-to-end system, significantly improving modeling success rate and reproducibility: This invention standardizes parameters for each step from sample pretreatment to drug sensitivity testing. Through an optimized Percoll density gradient centrifugation process, efficient enrichment of tumor cells and removal of impurities are achieved. The standardized operating manual effectively eliminates inter-laboratory operational errors, laying the foundation for large-scale clinical translation.

[0019] 2. The innovative culture system of this invention significantly enhances the proliferation and growth performance of organoids: Through systematic screening of growth factors and pleural effusion microenvironment components, this invention has determined the optimal culture medium formulation. Under this system, organoids proliferate significantly and reach structural maturity in just 7 days, significantly shortening the waiting period for clinical decision-making.

[0020] 3. This invention constructs a "three-in-one" multi-dimensional identification system to ensure the high fidelity of the model: Pathological fidelity: HE staining and detection of specific markers (CK7, TTF-1, etc.) ensure that organoids accurately reproduce the histological characteristics of the primary tumor.

[0021] Genetic consistency: Validated by whole-exome sequencing, lung cancer pleural effusion organoids and lung cancer pleural effusions showed extremely high consistency in terms of mutated gene landscape, somatic mutations, and fusion genes, fully replicating the core genetic characteristics of the original sample.

[0022] Functional correlation: By using 3D drug sensitivity testing and nAUC calculation, the in vitro drug response can be accurately mapped to the clinical efficacy, solving the problem of weak clinical correlation in existing models. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of an experimental procedure for the method in this invention; Figure 2 Bright-field photographs of the organoid model used in this invention; Figure 3 The image shows the HE staining and IHC identification results of lung cancer organoids in this invention. Figure 4 This is a diagram showing the mutation consistency analysis between lung cancer pleural effusion organoids and lung cancer pleural effusion in this invention; where A is a mutant gene landscape diagram, B is the somatic mutation consistency between pleural effusion PDO and pleural effusion, and C is the fusion gene consistency analysis. Figure 5 This is a bar chart showing the inhibition rates of different drug sensitivity regimens for lung cancer organoids in this invention; where a higher inhibition rate indicates stronger drug sensitivity. Figure 6 This is a dose-response diagram of drug sensitivity to lung cancer organoids for different drug sensitivity schemes in this invention. Detailed Implementation

[0024] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0025] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0026] This invention, through screening the ratios of various growth factors and inhibitors, finally established a complete culture medium formula. This formula, by synergistically regulating the Wnt, EGF and FGF signaling pathways and introducing autologous pleural effusion supernatant to compensate for microenvironmental factors, achieves rapid proliferation and high fidelity of lung cancer organoids.

[0027] This invention provides a systematic solution, including a standardized method for constructing organoids from lung cancer pleural effusion samples, a multidimensional identification system integrating pathology, genes, and function, and a drug sensitivity evaluation system. The specific technical solutions are described below: 1. Methods for isolating tumor cells from pleural effusion samples of lung cancer Using Percoll density gradient centrifugation, based on the inherent density differences among different cell types, this invention innovatively constructs a discontinuous dual-density gradient layer of 75% and 40%. Experiments have demonstrated that this specific gradient combination, under 800g centrifugal force, can precisely separate tumor cell clusters from red blood cells (sinking to the bottom) and light debris (top layer), enriching tumor cells in the junctional zone. This step is the core key to achieving a very high initial effective cell percentage.

[0028] 2. Conditions for organoid culture of tumors originating from pleural effusion in lung cancer The organoid basal medium was Advanced DMEM / F12, supplemented with the following components: 1×Glutamax supplement, 1×HEPES, 1×Penicillin-streptomycin, 1×B27 additive, 1×N2 additive, and various factors in the following concentration ranges: 1–100 mM Nicotinamide, 1–10 mM N-acetyl-L-cysteine, 1–1000 nM TGF-β receptor inhibitor (A83-01, i.e., TGF-β type I receptor inhibitor A83-01), 1–50 μM ROCK kinase inhibitor (Y27632), 1–100 ng / mL Human Noggin, 1–100 ng / mL Human Epidermal Growth Factor (EGF), and 1–500 ng / mL... Wnt-3a protein, 1~500 ng / mL R-spondin1 protein, and pleural effusion supernatant at a volume concentration of 1~100%.

[0029] The complete culture medium for lung cancer organoids in the drug sensitivity test contained the following components: Advanced DMEM / F12, 1× glutamine supplement, 1× hydroxyethylpiperazine ethanesulfonic acid, 1× penicillin-streptomycin, 1× B27 additive, 1× N2 additive, 10 mM nicotinamide, 1.25 mM N-acetyl-L-cysteine, 500 nM TGF-β receptor inhibitor, 10 μM ROCK kinase inhibitor, 25 ng / mL human recombinant Noggin protein, 25 ng / mL human epidermal growth factor, 100 ng / mL Wnt-3a protein, 250 ng / mL R-spondin1 protein, and pleural effusion supernatant at a final concentration of 10% by volume.

[0030] 3. Identification and Verification Protocol for Organoids Originating from Lung Cancer Pleural Effusion 1) Pathological verification HE staining: Organoids cultured for 7–10 days were fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin and sectioned, and stained with HE. Their pathological morphology and structure were observed under a microscope and compared with the patient's primary tumor tissue sections. Immunohistochemical (IHC) identification: Immunohistochemical techniques are used to detect the expression of lung cancer-related markers (such as CK7, TTF-1, Napsin A, CEA, etc.) in organoids to further confirm their tumor origin and phenotypic characteristics.

[0031] 2) Gene sequencing analysis Genomic DNA was extracted from organoids and corresponding pleural effusion samples. Whole-exome sequencing was used to systematically analyze gene mutation profiles, somatic cell mutations, and fusion genes to assess the consistency between organoid models and original pleural effusion samples at the level of gene variation.

[0032] 4. Drug sensitivity testing Organoids in good growth condition were harvested and exposed to 11 commonly used first- and second-line lung cancer treatment drugs, including gefitinib, osimertinib, and cisplatin, at different concentration gradients: single-drug settings (0 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM) and combination drug settings (0 μM, 0.0625 μM, 0.25 μM, 1 μM, 4 μM, 16 μM). After 6 days of treatment, cell viability was assessed using the CellCounting-Lite® 3D assay. The sensitivity of organoids to each drug was systematically evaluated by calculating the normalized area under the curve (nAUC) and the inhibition rate of the highest drug concentration.

[0033] Specifically, the relevant preparation and testing methods are as follows: This experimental procedure covers the entire process from lung cancer pleural effusion sample pretreatment, tumor cell isolation, organoid seeding and culture, to final sample collection and drug sensitivity testing, aiming to establish a standardized and reproducible organoid model construction and functional analysis system.

[0034] I. Experimental Procedure The construction process of organoids for lung cancer pleural effusion in this invention mainly includes key steps such as tumor cell isolation, organoid seeding and culture, pathological verification, genomic analysis, and drug sensitivity testing. A schematic diagram of the overall experimental process can be found here. Figure 1 .

[0035] 1. Pleural fluid sample pretreatment and cell separation This step aims to efficiently separate high-quality tumor cells from pleural fluid samples using the Percoll density gradient centrifugation method.

[0036] ① Take a sample of pleural effusion from human lung cancer, aliquot it into 50 mL centrifuge tubes, centrifuge at 500g for 5 min, discard the supernatant, and collect the cell pellet; ② Add an appropriate amount of tissue / organoid washing solution (DMEM medium, with 1× GlutaMAX, 1× HEPES, and 1× penicillin-streptomycin) to the cell pellet for resuspending. Filter using a 100 μm cell sieve, collect the filtrate in a centrifuge tube, centrifuge again, discard the supernatant, and add 5 mL of tissue / organoid washing solution to resuspend the cell pellet. ③ Resuspend the precipitate with 10 mL of 75% Percoll solution and transfer it to a new 50 mL centrifuge tube. Then slowly add 10 mL of 40% Percoll solution to the top layer to create a density gradient. ④ Slowly add a mixture of cells and tissue / organoid washing solution to the upper layer of a 40% Percoll solution. After centrifugation at 800g for 20 min, cells are separated into layers based on density differences. ⑤ Collect the top and middle layers rich in tumor cells, transfer them to a new 15 mL centrifuge tube, add 5 mL of tissue / organoid washing solution and mix well to obtain a cell suspension for later use.

[0037] 2. Organoid inoculation The 1×10 selected in this invention 5 Seeding cells at a concentration of 50 μL Matrigel gel per 24-well plate is the optimal solution that balances organoid modeling success rate and proliferation rate.

[0038] ① Take 30 μL of cell suspension and mix it with an equal volume of organoid fluorescent dye (AO / PI dual-color fluorescent dye, AO / PI staining solution), and count the cells using an organoid counter; ② Each 50 μL Matrigel gel contains 1×10 5 Prepare a mixture in proportion of individual cells or cell clusters, and inoculate it into a 24-well culture plate at a rate of 50 μL per well. ③ Incubate the culture plate in a 37℃, 5% CO2 incubator for 15 minutes to allow the Matrigel to solidify completely, thus creating a three-dimensional growth environment and promoting organoid formation.

[0039] 3. Organoid Culture ① Add 500 μL of complete lung cancer organoid culture medium to each well of the culture plate in steps 2 and ③ above; ② Continue to incubate the culture plate in a 37℃, 5% CO2 incubator; ③ Replace the culture medium with fresh one every 2–3 days, and regularly observe and record the growth status of organoids.

[0040] ④ In about 7-10 days, lung cancer organoids begin to form.

[0041] 4. Organoid sample collection ① Use pre-cooled tissue / organoid washing solution to release the organoids from the matrix gel by repeated blowing; ② Centrifuge and discard the supernatant, then wash with PBS to remove residual matrix gel; ③ Take approximately 5 × 10 5 Each organoid was added to 500 μL of tissue fixative for subsequent HE staining and IHC identification; ④ Take approximately 5 × 10 5 For each organoid, add 500 μL of RNAlater™ Stabilization (ThermoFisher SCIENTIFIC product) for genome sequencing analysis.

[0042] 5. Organoid drug sensitivity testing ① Select organoids in good growth condition, and use 500 organoids per well, 10% Matrigel gel and complete culture medium to make up a 50 μL system, and inoculate it into a 384-well plate; ② Place the culture plate in a 37℃, 5% CO2 incubator and incubate overnight for 1 day; ③ Design of drug treatment regimens: 11 drug sensitivity test regimens, including next-generation targeted drugs: osimertinib, dacomitinib, afatinib, amitinib, suvortinib, and icotinib mesylate (all of which are third-generation or higher EGFR-TKIs or novel irreversible inhibitors); early-stage targeted drugs: gefitinib, erlotinib, and icotinib (all of which are first-generation EGFR-TKIs); and chemotherapy drugs: pemetrexed + cisplatin and pemetrexed + carboplatin (both of which are commonly used combination chemotherapy regimens for lung cancer). For each drug, five concentration gradients (0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM) and one control group (0 μM) were prepared for single-drug therapy. For combination therapy, five concentration gradients (0.0625 μM, 0.25 μM, 1 μM, 4 μM, 16 μM) and one control group (0 μM) were prepared. Five replicates were added to each well in 50 μL of culture medium. Drug sensitivity testing was performed between 72 and 144 hours of culture. During this window period, the organoids are in the logarithmic growth phase and are most sensitive to drug response, which can most realistically reflect the cell behavior under clinical chemotherapy stress. ④ After adding the drug, place it in a 37℃, 5% CO2 incubator and continue culturing for 6 days; ⑤ Add 40 μL of CellCounting-Lite® 3D detection reagent to each well and incubate with a horizontal shaker for 10 minutes to ensure the reagent is fully utilized; ⑥ Use an enzyme-linked immunosorbent assay (ELISA) reader to detect organoid viability using chemiluminescence immunoassay; ⑦ Data analysis was performed using GraphPad Prism 8 software to assess drug sensitivity.

[0043] II. Test Results 1. Lung cancer pleural effusion samples were processed using Percoll density gradient centrifugation to establish a lung cancer organoid model. Bright-field photographs of the organoid model are shown below. Figure 2 As shown, from Figure 2 The results show that the organoids are plump, translucent, and numerous, with no obvious dead cells, indicating a high survival rate of tumor cells after treatment. After 7 days of culture, the average diameter of the organoids showed a significant increasing trend, demonstrating rapid growth characteristics. These results indicate that Percoll density gradient centrifugation can efficiently enrich tumor cells in lung cancer pleural effusion samples, providing a high-quality cell source for the stable construction of lung cancer organoids, and is a preferred technical solution for organoid modeling using lung cancer pleural effusion samples.

[0044] 2. Organoid identification Pathological verification results as follows Figure 3 As shown, HE staining revealed that the lung cancer organoids exhibited a typical acinar arrangement, significant cellular atypia, and a high nucleocytoplasmic ratio, completely consistent with the pathological morphology of primary lung cancer tissue. IHC (CK7, TTF-1, Napsin A, CEA) identification results showed that CK7, TTF-1, and Napsin A were all positively expressed in the organoids, confirming their tumorous nature. These results demonstrate that the lung cancer organoids constructed in this invention not only possess the typical pathological morphology of lung cancer tissue but also highly express lung cancer-specific markers, fully confirming their tumorous nature and consistency with the primary tumor.

[0045] 3. Whole exome sequencing analysis The results of whole-exome sequencing analysis are as follows: Figure 4As shown in Figure 4A, the left side shows the mutated gene landscape of pleural fluid samples (CJX_MPE, FCX_MPE), and the right side shows the mutated gene landscape of the corresponding pleural fluid organoids (CJX_PDO, FCX_PDO). It can be seen that the mutated gene types (including missense mutations, nonsense mutations, frameshift insertions / deletions, etc.), mutated gene distribution, and mutation frequency of the two groups of samples are highly overlapping. Moreover, 100% (4 / 4) of the high-frequency mutated genes (such as C>T, C>A, etc.) in the four samples are present in both pleural fluid and organoids, confirming that the mutated gene landscapes of the two are completely consistent. Figure 4B is a pie chart showing somatic mutation consistency. The somatic mutation consistency of the "CJX" group (CJX_MPE and CJX_PDO) reached 86%, and that of the "FCX" group (FCX_MPE and FCX_PDO) reached 89%. The overlap rate of mutation types (including synonymous mutations, missense mutations, etc.) and mutation sites in the two groups was ≥85%, with only ≤8% of organoid-specific low-frequency mutations (with no functional impact), fully demonstrating the high consistency of somatic mutations between the two groups. Figure 4C shows the consistency analysis of fusion genes (including the CJX and FCX groups). The fusion gene types of the pleural fluid samples (CJX_MPE, FCX_MPE) and their corresponding organoids (CJX_PDO, FCX_PDO) are completely matched: In the CJX group, the INV fusion of WDR7 and PIGN was detected in both; in the FCX group, the TRA fusion of RP11-40F8.2 and EEF1A2 was detected in both; confirming that the fusion genes of the two are basically consistent. Whole-exome sequencing results show that the organoids not only retain driver mutations such as EGFR, but also show high consistency (≥85%) in non-coding regions and fusion genes (such as WDR7-PIGN). This genetic 'clonal evolutionary stability' proves that the culture system of this invention can effectively inhibit the excessive growth of fibroblasts and maintain the purity of tumor cells.

[0046] The above results show that the pleural effusion organoid constructed in this invention has extremely high consistency with the original pleural effusion sample in terms of mutant gene landscape, somatic mutations and fusion genes, fully replicating the core genetic characteristics of the original sample, and providing key gene-level support for its reliability as a tumor model.

[0047] 4. Drug sensitivity testing The results are shown in Table 1.

[0048] Table 1. Statistical table of inhibition rates of different drug sensitivity regimens on lung cancer organoids.

[0049] Note: The inhibition rate of each group is the result of comparing the highest drug concentration group with the negative control group without drug (the inhibition rate of the control group is 0); different samples have different sensitivities to the drug, so a positive control could not be set up.

[0050] In this invention, the drug sensitivity testing method for lung cancer pleural effusion organoids is as follows: mature lung cancer pleural effusion organoids are cultured and inoculated into a suitable 3D culture system. Eleven drug regimens (including targeted drugs and chemotherapy drugs) are set up. Each group of single-drug and combination drugs has 5 concentration gradients and a negative control group (concentration of 0 μM). After culturing for 144 h, cell viability is detected by CellCounting-Lite® 3D method. The area under the normalized curve (nAUC) and the highest concentration inhibition rate are calculated. The lower the nAUC and the higher the inhibition rate, the stronger the sensitivity.

[0051] 1. Drug regimen and sensitivity parameter results (corresponding to Table 1) Table 1 shows the quantitative data of nAUC and inhibition rate of each group of drugs. The results show that: Next-generation targeted therapy group: nAUC ranged from 0.0755 (osimertinib) to 0.1236 (aflutinib mesylate), with a mean of ≤0.1; early-stage targeted therapy group: nAUC ranged from 0.4577 (gefitinib) to 0.5910 (icotinib), with a mean of ≥0.5; Chemotherapy group: nAUC ranged from 0.4420 (pemetrexed + cisplatin) to 0.4581 (pemetrexed + carboplatin), with a mean of ≥0.44. The nAUC data were consistent with the inhibition rate results, and the nAUC of the new generation targeted drug was significantly lower than that of the other two groups (difference ≥0.34), demonstrating a clear advantage in sensitivity.

[0052] 2. Results of the highest concentration inhibition rate (corresponding to) Figure 5 (bar chart) Figure 5 shows the distribution of the highest concentration inhibition rate of each drug group on organoids. The results show that: The next-generation targeted therapy group: osimertinib (regiment 1) achieved an inhibition rate of 97.38%, dacomitinib (regiment 2) 97.34%, afatinib (regiment 3) 94.91%, ametinib (regiment 4) 94.74%, suvortinib (regiment 5) 95.20%, and icotinib mesylate (regiment 6) 93.33%; the mean inhibition rate of this group was ≥93%. The early targeted therapy group: gefitinib (regiment 8) achieved an inhibition rate of 82.05%, erlotinib (regiment 10) 65.65%, and icotinib (regiment 11) only 53.49%; the mean inhibition rate of this group was ≤67%. The chemotherapy group: pemetrexed + cisplatin (regiment 7) achieved an inhibition rate of 65.99%, and pemetrexed + carboplatin (regiment 9) 67.28%; the mean inhibition rate of this group was ≤67%.

[0053] It is evident that the highest inhibition rate of the new generation of targeted drugs on organoids is significantly higher than that of the early targeted drug group (difference ≥26%) and the chemotherapy drug group (difference ≥26%).

[0054] The organoid model constructed in this invention accurately captures the efficacy ladder of different generations of targeted drugs. The extremely low nAUC values ​​(≤0.1) exhibited by third-generation drugs such as osimertinib are highly consistent with the high response rate of these drugs in advanced lung cancer in clinical practice, verifying the predictive reliability of this model as a "clinical substitute".

[0055] 3. Concentration-inhibition rate curve results (corresponding to...) Figure 6 (Dose-response relationship diagram) Figure 6 shows the concentration-inhibition rate curves for different drug groups. The results show that: The new generation of targeted drugs (bottom right curve): the curves of drugs such as osimertinib and amitinib decreased rapidly with increasing concentration, and the inhibition rate reached over 90% at 1 μM, with nAUC ≤ 0.12; the early targeted drugs (top right curve): the curves of drugs such as gefitinib and icotinib decreased more gradually, and the inhibition rate was still < 90% at 10 μM, with nAUC ≥ 0.45; the chemotherapy drugs (bottom left curve): the curve of the pemetrexed combination regimen decreased the least, with an inhibition rate of only about 70% at 10 μM, with nAUC ≥ 0.44.

[0056] The above results confirm that the lung cancer pleural effusion organoid constructed in this invention can accurately distinguish the differences in sensitivity of different drugs: the inhibitory activity of the new generation of targeted drugs is significantly better than that of early targeted drugs and chemotherapy drugs, which fully demonstrates the accuracy and reliability of this organoid model in the individualized drug screening of lung cancer, and provides an efficient and controllable in vitro evaluation tool for the optimization of clinical medication regimens.

[0057] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A standardized method for constructing lung cancer pleural effusion organoids with high clinical consistency, characterized in that: Includes the following steps: (1) Cell separation: The lung cancer pleural fluid sample was subjected to density gradient centrifugation. The centrifugation used a dual-concentration Percoll layer with a bottom layer of 75% mass concentration and a middle layer of 40% mass concentration. The mixture was centrifuged at 800g for 20min and the cell layer at the interface between the two Percoll layers was collected. (2) Inoculation: The collected cells were inoculated at a rate of 1×10⁻⁶. 5 -5×10 5 Inoculate the culture plate at a density of 1 per 50 μL of matrix gel and incubate until solidification. (3) Culture: Cultured using a complete culture medium, which includes basal culture medium, B27, N2, nicotinamide, N-acetyl-L-cysteine, A83-01, Y27632, Noggin, EGF, Wnt-3a, R-spondin1, FGF7, FGF10 and lung cancer pleural effusion supernatant with a volume fraction of 5%-20%; (4) Establish an organoid model of pleural effusion in lung cancer; (5) Multidimensional identification: HE staining pathological identification, IHC marker identification and whole exon sequencing verification were performed on the cultured organoids. The verification was successful, and lung cancer pleural effusion organoids with high clinical consistency were obtained.

2. The construction method according to claim 1, characterized in that: The specific steps of step (1) are as follows: ① Take human lung cancer pleural effusion samples, aliquot them into 50 mL centrifuge tubes, centrifuge at 500g for 5 min, discard the supernatant, and collect the cell pellet; ② Add tissue / organoid washing solution to the cell pellet for resuspending, filter using a 100 μm cell sieve, collect the filtrate in a centrifuge tube, centrifuge again at 500g for 5 min, discard the supernatant and add 5 mL of tissue / organoid washing solution to resuspend the cell pellet. ③ Resuspend the precipitate with 10 mL of 75% Percoll solution and transfer it to a new 50 mL centrifuge tube. Then slowly add 10 mL of 40% Percoll solution to the top layer to create a density gradient. ④ Slowly add a mixture of cells and tissue / organoid washing solution to the upper layer of a 40% Percoll solution. After centrifugation at 800g for 20 min, cells are separated into layers based on density differences. ⑤ Collect the top and middle layers rich in tumor cells, transfer them to a new 15 mL centrifuge tube, add 5 mL of tissue / organoid washing solution and mix well to obtain a cell suspension for later use.

3. The construction method according to claim 2, characterized in that: The formulation of the tissue / organoid washing solution in step ② includes: 1× GlutaMAX, 1× HEPES, and 1× penicillin-streptomycin added to DMEM medium.

4. The construction method according to claim 1, characterized in that: The specific steps of step (2) are as follows: ① Take 30 μL of cell suspension and mix it with an equal volume of organoid fluorescent dye, then use an organoid counter to count the cells; ② Each 50 μL Matrigel gel contains 1×10 5 A mixture was prepared in a ratio of individual cells to cell clusters and seeded into 24-well culture plates at 50 μL per well. ③ Incubate the culture plate in a 37℃, 5% CO2 incubator for 15 minutes to allow the Matrigel to solidify completely, thereby creating a three-dimensional growth environment and promoting organoid formation; The specific steps of step (3) are as follows: Organoid culture: ① Add 500 μL of complete lung cancer organoid culture medium to each well of the culture plate in step (2) ③ above; ② Continue to incubate the culture plate in a 37℃, 5% CO2 incubator; ③ Replace the culture medium with fresh one every 2–3 days, and observe and record the growth status of organoids regularly.

5. The construction method according to claim 4, characterized in that: The formulation of the complete lung cancer organoid culture medium is as follows: The organoid basal medium was Advanced DMEM / F12, containing the following supplementary components: 1× glutamine supplement, 1× hydroxyethylpiperazine ethanesulfonic acid, 1× penicillin-streptomycin, 1× B27 additive, 1× N2 additive, and various factors in the following concentration ranges: 1~100 mM nicotinamide, 1~10 mM N-acetyl-L-cysteine, 1~1000 nM TGF-β receptor inhibitor, 1~50 μM ROCK kinase inhibitor, 1~100 ng / mL human recombinant Noggin protein, 1~100 ng / mL human epidermal growth factor, 1~500 ng / mL Wnt-3a protein, 1~500 ng / mL R-spondin1 protein, and pleural effusion supernatant at 1~100% volume concentration.

6. The construction method according to claim 4, characterized in that: The formulation of the complete lung cancer organoid culture medium is as follows: The basal culture medium consisted of Advanced DMEM / F12, 1× glutamine supplement, 1× hydroxyethylpiperazine ethanesulfonic acid, 1× penicillin-streptomycin, 1× B27 additive, 1× N2 additive, 10 mM nicotinamide, 1.25 mM N-acetyl-L-cysteine, 500 nM TGF-β receptor inhibitor, 10 μM ROCK kinase inhibitor, 25 ng / mL human recombinant Noggin protein, 25 ng / mL human epidermal growth factor, 100 ng / mL Wnt-3a protein, 250 ng / mL R-spondin1 protein, and pleural effusion supernatant at a final volume concentration of 10%.

7. The construction method according to claim 1, characterized in that: The specific steps of step (4) are as follows: ① Use pre-cooled tissue / organoid washing solution to release the organoids from the matrix gel by repeated blowing; ② Centrifuge and discard the supernatant, then wash with PBS to remove residual matrix gel; Alternatively, the specific steps of step (5) are as follows: 1) Take 5 × 10 5 Each organoid was treated with 500 μL of tissue fixative (4% paraformaldehyde) for subsequent HE staining and IHC identification. 2) Take 5 × 10 5 Each organoid was added to 500 μL of RNA stabilization solution for genome sequencing analysis.

8. A lung cancer pleural effusion organoid with high clinical consistency constructed by the construction method according to any one of claims 1 to 7.

9. The application of lung cancer pleural effusion organoids as described in claim 8 in personalized drug screening for lung cancer.

10. The method for screening personalized medicines for lung cancer using organoids derived from pleural effusion as described in claim 8, characterized in that: Including the following methods: ① Select organoids, and use 500 organoids per well to make up a 50 μL system with 5-15% Matrigel gel and complete culture medium, and seed it in a 384-well plate; ② Place the culture plate in a 37℃, 5% CO2 incubator and incubate overnight for 1 day; ③ Design drug treatment protocols: Design drug sensitivity protocols, with five concentration gradients for each drug as a single agent (0.01 μM, 0.1 μM, 1 μM, 10 μM, 100 μM) and one control group (0 μM). For combination drugs, five concentration gradients are set up (0.0625 μM, 0.25 μM, 1 μM, 4 μM, 16 μM) and one control group (0 μM). Each condition is prepared in 5 replicates and added to 50 μL of culture system. Drug sensitivity testing is performed from 72h to 144h of culture. During this window period, organoids are in the logarithmic growth phase and are most sensitive to drugs, which can most realistically reflect the cell behavior under clinical chemotherapy stress. ④ After adding the drug, place it in a 37℃, 5% CO2 incubator and continue culturing for 6 days; ⑤ Add 40 μL of detection reagent to each well and incubate with a horizontal shaker for 10 minutes to ensure the reagent is fully reacted; ⑥ Use an enzyme-linked immunosorbent assay (ELISA) reader to detect organoid viability using chemiluminescence immunoassay; ⑦ Conduct data analysis to assess drug sensitivity.

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