Method for establishing lung cancer 2.5 D organ phenotype plasticity transformation model

Abstract

The invention provides an establishment method of a lung cancer 2.5 D organ phenotype plasticity transformation model. The establishment method comprises the following steps: step S1, obtaining a malignant pleural effusion sample of a lung cancer patient; s2, performing 2.5 D organoid culture on the sample to form a suspended phenotype organoid; s3, converting the organoid from a suspension phenotype to a wall-attaching phenotype; S4, converting the organoid from the wall-attaching phenotype to the suspension phenotype; s5, repeating the step S3 and the step S4 to realize multiple cycles of phenotypic transformation; and S6, identifying genetic feature differences. The invention relates to the technical field of organoid, and successfully establishes a lung cancer 2.5 D organoid phenotype plasticity transformation model, the model overcomes a plurality of defects of a traditional in-vitro model, can more truly simulate phenotype change of lung cancer cells in an in-vivo microenvironment, and has a good application prospect. And an innovative and effective research platform is provided for researching the drug resistance and metastasis mechanism driven by the phenotype plasticity of the tumor cells.

Description

Technical Field

[0001] This invention relates to the field of organoid technology, and in particular to a method for establishing a 2.5D organoid phenotypic plasticity transformation model for lung cancer. Background Technology

[0002] Lung cancer is one of the leading causes of cancer-related morbidity and mortality worldwide, encompassing two major histological subtypes: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). Advanced-stage patients often present with malignant serous cavity effusions (such as pleural effusion and pericardial effusion), resulting in extremely poor prognosis. Although targeted therapy and chemotherapy can prolong the survival of some patients, tumor heterogeneity and treatment resistance remain major clinical challenges. Among these challenges, tumor cell phenotypic plasticity (the ability of cancer cells to adapt to their microenvironment and alter their biological characteristics) is a key driver of drug resistance and metastasis, and this plasticity is also one of the reasons for the high heterogeneity of lung cancer.

[0003] In vitro models are of great significance in lung cancer research and treatment. Traditional in vitro models, such as two-dimensional cell culture, while simple to operate and low in cost, cannot realistically simulate the biological behavior of lung cancer in vivo due to the lack of complex three-dimensional structures and intercellular interactions. Related studies have shown that cell growth, differentiation, and drug response in two-dimensional cell culture environments deviate significantly from actual in vivo conditions, severely limiting their ability to predict clinical treatment outcomes. While patient-derived xenograft models in animal models can reflect the histopathological and genetic expression characteristics of the primary tumor to some extent, their construction is fraught with difficulties. For lung cancer, the success rate of model formation using endobronchial ultrasound-guided transbronchial needle aspiration biopsy and computed tomography-guided biopsy samples is only 26%-47.1%; the modeling cycle often takes months or even longer; and the cost is high, requiring significant human, material, and financial resources. These problems greatly limit the application of traditional in vitro models in personalized medicine for lung cancer, failing to meet the urgent clinical need for accurate prediction of treatment outcomes and the development of personalized treatment plans.

[0004] While patient-derived organoid models can preserve the histopathological and genomic characteristics of parental tumors to some extent, and their overall accuracy in predicting clinical efficacy using drug sensitivity testing reaches 83.3%, existing organoid models primarily focus on drug sensitivity testing, and their simulation of tumor cell phenotypic plasticity induced by the microenvironment remains incomplete. Therefore, establishing a lung cancer organoid transformation model that can simulate tumor phenotypic plasticity is of great significance for elucidating drug resistance mechanisms, developing combination therapies, and promoting precision medicine. Summary of the Invention

[0005] In view of this, the present invention aims to propose a method for establishing a 2.5D organoid phenotypic plasticity transformation model for lung cancer, which provides a reliable tool for in-depth research on the drug resistance mechanism of lung cancer and has important application value and broad prospects in developing combination therapies and realizing personalized precision medicine for lung cancer.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0007] A method for establishing a 2.5D organoid phenotypic plasticity transformation model for lung cancer includes the following steps: Step S1: Obtain malignant pleural effusion samples from lung cancer patients. The samples must meet the following criteria: patient age 18-65 years, tumor cell count ≥10 6 Patients with multiple cancers, those currently receiving targeted therapy, chemotherapy, or intrapleural chemotherapy, and those whose lung adenocarcinoma has transformed into squamous cell carcinoma or small cell lung cancer are excluded.

[0008] Step S2: Culture the sample into 2.5D organoids to form suspension phenotype organoids;

[0009] Step S3: Transformation of organoids from suspension phenotype to adherent phenotype: Transfer the suspension organoids into a 10cm cell culture dish, add 10ml of DMEM-F12 culture medium containing antibiotics and 10% fetal bovine serum, and incubate at 37℃, 5% CO2 for 2-3 days until the organoids adhere to the wall and form "hills". Continue culturing for 2-3 days until the "hills" flatten and cover the entire culture dish. After digesting and recovering the cells with trypsin, re-seed the cells to obtain adherent phenotype cells.

[0010] Step S4: Organoids are transformed from adherent phenotype to suspension phenotype: The adherent cells obtained in step S3 are cultured until the semi-suspended and semi-adherent cells proliferate. The medium is then replaced with a semi-solid medium and cultured for 3-5 days until the cells aggregate into spheres to form suspension phenotype organoids.

[0011] Step S5: Repeat steps S3 and S4 to perform multiple loops of phenotypic transformation;

[0012] Step S6: When the maximum diameter of the organoids in step S5 is greater than 50 μm, the organoid cell clusters are recovered using a 40 μm cell sieve and centrifuged at 200g for 5 minutes to collect the organoid precipitate; then, phenotypic plasticity models are established for organoids with diameters greater than 40 μm and less than 40 μm respectively according to step S5.

[0013] Furthermore, the lung cancer type is non-small cell lung cancer, and the pancreatic enzyme digestion conditions in step S3 are 0.25% concentration, 37°C digestion for 2 minutes.

[0014] Furthermore, the lung cancer type is small cell lung cancer, and the pancreatic enzyme digestion conditions in step S3 are 0.25% concentration, 37°C digestion for 1 minute, and the operation can be repeated.

[0015] Furthermore, in step S2, suspended organoids cultured for 6-12 days are preferentially selected for phenotypic transformation.

[0016] Furthermore, the semi-solid culture medium described in step S4 consists of pleural effusion supernatant and DMEM-F12 culture medium containing 10% fetal bovine serum.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] The method for establishing a 2.5D organoid phenotypic plasticity transformation model for lung cancer described in this invention has successfully established a 2.5D organoid phenotypic plasticity transformation model for lung cancer, covering both non-small cell lung cancer and small cell lung cancer. Through screening and processing of pleural effusion samples from patients, combined with specific culture and operational procedures, the model achieves stable and reversible transformation of organoids between suspension and adherent phenotypes, and maintains phenotypic plasticity characteristics even after multiple cycles. This model overcomes many shortcomings of traditional in vitro models, and can more realistically simulate the phenotypic changes of lung cancer cells in the in vivo microenvironment, providing an innovative and effective research platform for studying the drug resistance and metastasis mechanisms driven by tumor cell phenotypic plasticity. Attached Figure Description

[0019] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0020] Figure 1 This invention relates to a method for establishing a 2.5D organoid phenotypic plasticity transformation model for lung cancer, as described in this embodiment, and to identifying phenotypic plasticity models and genetic differences in 2.5D organoids for non-small cell lung cancer.

[0021] Figure 2 This invention relates to a method for establishing a 2.5D organoid phenotypic plasticity transformation model for lung cancer, as described in this embodiment, and to identifying phenotypic plasticity models and genetic differences in 2.5D organoids for small cell lung cancer.

[0022] Figure 3 The diameter of small cell lung cancer 2.5D organoids affects their phenotypic transformation efficiency in the method for establishing a phenotypic plasticity transformation model of lung cancer 2.5D organoids as described in the embodiments of the present invention. Detailed Implementation

[0023] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0024] In the description of this invention, it should be noted that the terms "upper," "lower," "inner," and "back," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0025] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0026] This embodiment relates to a method for establishing a 2.5D organoid phenotypic plasticity transformation model for lung cancer, such as... Figures 1-3 As shown, it includes the following steps:

[0027] Step S1: Obtain malignant pleural effusion samples from lung cancer patients. The samples must meet the following criteria: patient age 18-65 years, tumor cell count ≥10. 6 Patients with multiple cancers, those currently receiving targeted therapy, chemotherapy, or intrapleural chemotherapy, and those whose lung adenocarcinoma has transformed into squamous cell carcinoma or small cell lung cancer are excluded.

[0028] Step S2: Culture the sample into 2.5D organoids to form suspension phenotype organoids;

[0029] Step S3: Transformation of organoids from suspension phenotype to adherent phenotype: Transfer the suspension organoids into a 10cm cell culture dish, add 10ml of DMEM-F12 culture medium containing antibiotics and 10% fetal bovine serum, and incubate at 37℃, 5% CO2 for 2-3 days until the organoids adhere to the wall and form "hills". Continue culturing for 2-3 days until the "hills" flatten and cover the entire culture dish. After digesting and recovering the cells with trypsin, re-seed the cells to obtain adherent phenotype cells.

[0030] Step S4: Organoids are transformed from adherent phenotype to suspension phenotype: The adherent cells obtained in step S3 are cultured until the semi-suspended and semi-adherent cells proliferate. The medium is then replaced with a semi-solid medium and cultured for 3-5 days until the cells aggregate into spheres to form suspension phenotype organoids.

[0031] Step S5: Repeat steps S3 and S4 to perform multiple loops of phenotypic transformation;

[0032] Step S6: When the maximum diameter of the organoids in step S5 is greater than 50 μm, the organoid cell clusters are recovered using a 40 μm cell sieve and centrifuged at 200g for 5 minutes to collect the organoid precipitate; then, phenotypic plasticity models are established for organoids with diameters greater than 40 μm and less than 40 μm respectively according to step S5.

[0033] The lung cancer type is non-small cell lung cancer. In step S3, the pancreatic enzyme digestion conditions are 0.25% concentration and 37°C for 2 minutes.

[0034] The lung cancer type is small cell lung cancer. In step S3, the pancreatic enzyme digestion conditions are 0.25% concentration, 37°C digestion for 1 minute, and the operation can be repeated.

[0035] In step S2, suspended organoids cultured for 6-12 days are preferred for phenotypic transformation.

[0036] In step S4, the semi-solid culture medium consists of pleural effusion supernatant and DMEM-F12 medium containing 10% fetal bovine serum.

[0037] Specifically, sample acquisition in step S1 involves collecting malignant pleural effusion samples from pathologically confirmed lung cancer patients aged 18-65 years, including both non-small cell lung cancer and small cell lung cancer. The pleural effusion should be 200ml, and the tumor cell count should be on the order of 10-1. 6 More than one organoid model can be used for the establishment of this study. Patients with multiple cancers, those undergoing targeted therapy, chemotherapy, or intrapleural chemotherapy, and those whose lung adenocarcinoma has transformed into squamous cell carcinoma or small cell lung cancer should be excluded from this study. Written informed consent has been obtained from patients for this study, and it has been approved by the Ethics Committee of Jilin Provincial Cancer Hospital (Approval No.: 202306-023-01).

[0038] The 2.5D organoid culture method in step S2 is carried out in accordance with application number: 202411110546.2.

[0039] Based on the foregoing, the process of establishing the phenotypic plasticity model of 2.5D organoids from non-small cell lung cancer is as follows:

[0040] The organoids were transferred into 10cm cell culture dishes, and 10ml of DMEM-F12 culture medium containing penicillin antibiotics and 10% fetal bovine serum was added. The dishes were then incubated at 37°C in a 5% CO2 incubator for 2-3 days. The organoids adhered to the culture dish and grew, forming "hills". After culturing for another 2-3 days, new cells slowly emerged from around the organoids, and the "hills" gradually flattened. Once the cells had filled the culture dish, they were digested with 0.25% trypsin at 37°C for 2 minutes, centrifuged at 200g for 5 minutes to recover the cell suspension, and then re-seeded into new cell culture dishes. Once the cells re-adhere to the culture dish, the transformation of the organoids from suspension to adherent phenotype was completed. Continue culturing the adherent cells until they cover the entire bottom of the cell plate. Once the number of semi-suspended, semi-adherent cells gradually increases, change the cell culture medium and continue culturing for 3-5 days. The semi-suspended, semi-adherent cells will gradually aggregate into spheres. Once the diameter of the cell spheres is greater than 20 μm, change the 2.5D organoid culture medium mentioned above and continue culturing for 2-3 days. Organoids can be reformed, meaning that the organoids have completed the transformation from an adherent phenotype to a suspension phenotype.

[0041] The process of establishing a phenotypic plasticity model for 2.5D organoids of small cell lung cancer is as follows:

[0042] Tumor cells were resuspended in DMEM-F12 medium and counted at 5 x 10⁻⁶ cells / day. 7 Tumor cells were resuspended in pleural effusion supernatant and cultured in a 37°C, 5% CO2 incubator. After 3-5 days, the cells aggregated and were replaced with DMEM-F12 medium containing 10% fetal bovine serum and cultured for another 3-7 days. Once the cells clump together to form organoids, semi-suspended semi-adherent cells, and adherent cells, the suspended organoids were aspirated and cultured separately. These organoids are the organoid models with a suspension phenotype. The suspension-type organoids were transferred into 10cm flat-bottomed cell culture dishes, and 10 ml of DMEM-F12 culture medium containing 10% FBS was added. The dishes were then incubated in a 5% CO2 incubator for 2-3 days. The organoids gradually adhered to the culture dish and grew, forming "hills". After culturing for another 2-3 days, new cells slowly emerged from around the organoids, and the "hills" gradually flattened. Once the cells had filled the cell culture dish, they were digested with 0.25% trypsin at 37°C for 1 minute. If the digestion was incomplete, it could be repeated. The cells were then centrifuged at 200g for 5 minutes to recover the cell suspension and re-seeded into new cell culture dishes. Once the cells re-adhered to the culture dish, the transformation of the organoids from suspension to adherent phenotype was completed, thus completing the organoid suspension-to-adherent transformation model. The adherent cells were cultured further until they covered the entire bottom of the cell culture dish. Once the number of semi-suspended, semi-adherent cells gradually increased, the culture medium was replaced with the semi-solid medium used in the 2.5D organoid model construction described above. Cultured for another 3-5 days, until the semi-suspended, semi-adherent cells gradually aggregated into spheres, signifying the transformation from an adherent phenotype to a suspension phenotype. This method allows for repeated induction of phenotypic transformation, achieving phenotypic plasticity in small cell lung cancer.

[0043] Specifically, step S6, which identifies genetic traits, is as follows:

[0044] Once the maximum diameter of the organoids in step S5 exceeds 50 μm, the organoid cell clusters are recovered using a 40 μm cell sieve, and the organoid precipitate is collected by centrifugation at 200g for 5 minutes. Then, phenotypic plasticity models are established for organoids with diameters greater than 40 μm and less than 40 μm, respectively, following step S5.

[0045] A phenotypic plasticity model of 2.5D organoids from non-small cell lung cancer was established. After 3-5 days of culture, tumor cells formed suspension organoids with an average diameter of approximately 30-100 μm. After 5-10 days of culture, these organoids spontaneously differentiated into mature organoids with an average diameter of approximately 50-100 μm, possessing epithelial and stromal structures. Simultaneously, the bottom of the culture dish was covered with adherent cells and semi-suspended / semi-adherent cells. After separating and culturing the suspension organoids from the underlying cells, it was observed that the suspension organoids gradually adhered to the culture dish within 2-3 days, extending pseudopodia and crawling out of surrounding cells, forming a "hill-like" protruding adherent morphology. Figure 1 As shown in AB; after 5-8 days, the "hill" morphology disappeared, and the phenotype completely transformed into adherent cells and semi-suspended semi-adherent cells, as shown in AB. Figure 1 As shown in C. The semi-suspended, semi-adherent cells at the bottom layer after organoid removal proliferate significantly within 3-5 days, as shown in Figure C. Figure 1 As shown in DE, the cells cover the bottom of the culture dish and gradually re-aggregate into initial suspended organoid spheres, completing the transformation of cell phenotype. Figure 1 As shown in F. These results suggest that this study successfully constructed organoids derived from malignant pleural effusion in non-small cell lung cancer and successfully explored a method for constructing a phenotypic plasticity model that induces organoid phenotypes to change from suspension to adherence and from adherence to suspension.

[0046] Specifically, Figure 1 A: Suspended organoids extend pseudopodia to become adherent. B: New cells emerge from the "hill"-like structures surrounding the organoids. C: The "hill"-like cells complete the transformation into adherent cells and semi-suspended / semi-adherent cells. D: Adherent cells at the bottom of the petri dish after removing suspended organoids. E: Semi-suspended / semi-adherent cells proliferate in large numbers. F: Primary organoid spheres begin to aggregate and complete the transformation from adherent cells to suspended organoid phenotypes. G: Genetic differences among organoids with different phenotypes are analyzed through gene sequencing. Scale bar is 50 μm.

[0047] To establish a phenotypic plasticity model for 2.5D organoids of small cell lung cancer, the optimal state for suspension organoids cultured within 6-12 days is selected. For example... Figure 2 As shown in Figure AB, tumor cells in malignant pleural effusion can form suspended organoids with a diameter of approximately 40-100 μm. When these organoids are isolated and cultured individually in a petri dish, they gradually adhere to the dish within 48 hours, forming a hill-like, protruding morphology. Figure 2 As shown in C, if the adherent organoids are cultured further, their area will increase due to cell proliferation. Figure 2 As shown in D, within 72 hours, the cell phenotype transforms into adherent cells and semi-suspended / semi-adherent cells, which will cover the entire bottom of the petri dish as shown. Figure 2 As shown in E, the transformation of organoids from a suspended phenotype to an adherent phenotype is completed, marking the completion of the establishment of the adherent phenotype organoid model.

[0048] Specifically, Figure 2 A: Organoids with a suspension phenotype; B: Magnified organoids; C: Conversion from a suspension phenotype to an adherent phenotype; D: Cell proliferation leading to an increase in the area of ​​adherent cells; E: Adherent cells and semi-suspended / semi-adherent cells covering the bottom of the culture dish; F: Conversion from an adherent phenotype back to a suspension phenotype; G: Genetic differences in organoids with different phenotypes analyzed by gene sequencing. Scale bar = 100 μm.

[0049] After digesting the adherent cells with 0.25% trypsin, they were re-seeded into culture dishes. Following washing away the non-adherent suspension with PBS buffer, the adherent and semi-suspended / semi-adherent cells at the bottom of the dish proliferated significantly within 2-5 days. The semi-suspended / semi-adherent cells then developed into suspension-type organoids within 3-5 days. Figure 2 As shown in F, the transformation of organoids from adherent to suspended phenotypes has been completed, marking the successful establishment of the suspended phenotype organoid model. Furthermore, compared to organoids with a diameter less than 40 μm, 42.3 ± 7.5%, such as... Figure 3 As shown in Figure A, organoids larger than 40 μm accounted for 65.8 ± 10.9%, such as... Figure 3 As shown in B, the resulting phenotypic transformation model is more efficient (p<0.05).

[0050] Specifically, Figure 3 In section A: Organoids with a diameter less than 40 μm transition from an adherent phenotype to a suspension phenotype. In section B: Organoids with a diameter greater than or equal to 40 μm transition from an adherent phenotype to a suspension phenotype. Scale bar = 100 μm.

[0051] This phenotypic transformation can occur repeatedly, marking the successful establishment of a small cell lung cancer organoid model with phenotypic plasticity.

[0052] This invention successfully established a 2.5D organoid phenotypic plasticity transformation model for lung cancer, covering both non-small cell lung cancer and small cell lung cancer. Through screening and processing of pleural effusion samples from patients, combined with specific culture and operational procedures, the model achieved stable and reversible transformation of organoids between suspension and adherent phenotypes, with a genetic trait difference rate of <5% between phenotypes, exhibiting high fidelity and maintaining phenotypic plasticity characteristics even after multiple cycles. This model overcomes many shortcomings of traditional in vitro models, more realistically simulating phenotypic changes in lung cancer cells within the in vivo microenvironment, and providing an innovative and effective research platform for studying drug resistance and metastasis mechanisms driven by tumor cell phenotypic plasticity.

[0053] In practical applications, this model can help researchers delve into the molecular mechanisms of drug resistance in lung cancer, thereby enabling the targeted development of novel combination therapies and improving the treatment outcomes of lung cancer. Simultaneously, based on the model's simulation of tumor cell phenotypic plasticity, it holds promise for accurately predicting treatment outcomes in lung cancer patients, providing crucial evidence for developing personalized treatment plans in clinical practice, and promoting the development of precision medicine for lung cancer.

[0054] Furthermore, this invention reveals that organoid diameter affects the phenotypic transformation efficiency of small cell lung cancer, providing direction for subsequent model optimization and improved modeling efficiency. In the future, further in-depth research into the model's biological characteristics can expand its application in basic research and clinical practice of lung cancer, such as exploring the impact of different therapeutic drugs on the phenotypic plasticity of tumor cells in the model, and applying the model to the evaluation of new lung cancer treatment strategies, contributing to overcoming this major challenge of lung cancer.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for establishing a 2.5D organoid phenotypic plasticity transformation model for lung cancer, characterized in that, Includes the following steps: Step S1: Obtain malignant pleural effusion samples from lung cancer patients. The samples must meet the following criteria: patient age 18-65 years, tumor cell count ≥10. 6 Patients with multiple cancers, those currently receiving targeted therapy, chemotherapy, or intrapleural chemotherapy, and those whose lung adenocarcinoma has transformed into squamous cell carcinoma or small cell lung cancer are excluded. Step S2: Culture the sample into 2.5D organoids to form suspension phenotype organoids; Step S3: Transformation of organoids from suspension phenotype to adherent phenotype: Transfer the suspension organoids into a 10cm cell culture dish, add 10ml of DMEM-F12 culture medium containing antibiotics and 10% fetal bovine serum, and incubate at 37℃ and 5% CO2 for 2-3 days until the organoids adhere to the wall and form "hills". Continue culturing for 2-3 days until the "hills" flatten and cover the entire culture dish. After digesting and recovering the cells with trypsin, re-seed the cells to obtain adherent phenotype cells. Step S4: Organoids are transformed from adherent phenotype to suspension phenotype: The adherent cells obtained in step S3 are cultured until the semi-suspended and semi-adherent cells proliferate. The medium is then replaced with a semi-solid medium and cultured for 3-5 days until the cells aggregate into spheres to form suspension phenotype organoids. Step S5: Repeat steps S3 and S4 to perform multiple loops of phenotypic transformation; Step S6: When the maximum diameter of the organoids in step S5 is greater than 50 μm, the organoid cell clusters are recovered using a 40 μm cell sieve and centrifuged at 200g for 5 minutes to collect the organoid precipitate; then, phenotypic plasticity models are established for organoids with diameters greater than 40 μm and less than 40 μm respectively according to step S5.

2. The method for establishing a 2.5D organoid phenotypic plasticity transformation model for lung cancer according to claim 1, characterized in that: The lung cancer type is non-small cell lung cancer, and the pancreatic enzyme digestion conditions in step S3 are 0.25% concentration, 37°C digestion for 2 minutes.

3. The method for establishing a 2.5D organoid phenotypic plasticity transformation model for lung cancer according to claim 1, characterized in that: The lung cancer type is small cell lung cancer. In step S3, the pancreatic enzyme digestion conditions are 0.25% concentration, 37°C digestion for 1 minute, and the operation can be repeated.

4. The method for establishing a 2.5D organoid phenotypic plasticity transformation model for lung cancer according to claim 3, characterized in that: In step S2, suspended organoids cultured for 6-12 days are preferred for phenotypic transformation.

5. The method for establishing a 2.5D organoid phenotypic plasticity transformation model for lung cancer according to claim 1, characterized in that: The semi-solid culture medium described in step S4 consists of pleural effusion supernatant and DMEM-F12 culture medium containing 10% fetal bovine serum.

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