Lung cancer organoid verification and function identification technological process

Abstract

The invention provides a lung cancer organoid verification and function identification technological process which comprises the following steps: step 1, collecting organoid, and step 2, carrying out verification and function identification on the organoid in the step 1. Wherein the verification and function identification in the step 2 are as follows: evaluating a development state; evaluating tumor characteristics; evaluating physiological functions; biological functions; identifying genetic characteristics; evaluating an immune microenvironment; and evaluating the drug sensitivity. The invention relates to the technical field of biomedicine, and provides a lung cancer organoid verification and function identification process flow, which aims to improve the quality of an organoid model, limit the application range of the organoid and finally provide help for exerting correct clinical and scientific research application values according to the performance of the organoid.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and particularly to a process flow for the verification and functional identification of lung cancer organoids. Background Art

[0002] The field of lung cancer treatment and research is standing at the new frontier of precision medicine, and there is an urgent need for the progress of innovative technologies to improve the treatment effect and patient survival rate. Organoid technology has become a research hotspot due to its unique ability to simulate the microenvironment and biological characteristics of tumors, providing a powerful platform for oncology research to construct disease models, screen drugs, and develop personalized treatment strategies. Although organoid technology shows great application potential, its wide adoption in clinical applications is limited by the lack of standardized verification and functional identification processes. Existing verification and identification methods, such as morphological evaluation and immunohistochemistry, although they can provide basic information, have deficiencies in accuracy and repeatability. At the same time, although next-generation sequencing (NGS) and RNA sequencing (RNA-seq) technologies can reveal deeper molecular information, the application of these technologies in the verification of lung cancer organoids has not yet formed a unified standard process. There are various existing verification and identification methods for organoids, and many researchers only identify from a single perspective, lacking a comprehensive understanding and quality control of organoids. Currently, there is no mature all-round verification and identification process flow.

[0003] To overcome this challenge, it is particularly crucial to establish a standardized verification and functional identification process flow for lung cancer organoids. Such a standardized process can not only enhance the reliability and effectiveness of the organoid model but also promote its application in drug research and development and personalized medicine. In addition, the standardized process also plays an important role in promoting the development of precision lung cancer treatment. It can help researchers better understand the heterogeneity and microenvironment of tumors, provide more precise treatment strategies for patients, reduce side effects, and thus enable patients to benefit more from treatment.

[0004] In summary, the precision treatment of lung cancer requires a model that highly simulates tumor characteristics. Organoid technology has attracted much attention because it can retain tumor heterogeneity and microenvironment, but the existing methods lack a systematic verification process, resulting in uneven quality of organoids and affecting downstream applications. Existing technologies mostly rely on a single indicator (such as morphology or some molecular markers) and cannot comprehensively evaluate the physiological functions, genetic characteristics, and drug sensitivities of organoids. Therefore, a standardized multi-dimensional verification process is urgently needed. Summary of the Invention

[0005] In view of this, the present invention aims to propose a process flow for the verification and functional identification of lung cancer organoids to ensure that they possess various physiological and biological functions of lung cancer tumors, thereby forming a standardized lung cancer organoid model.

[0006] To achieve the above object, the technical solution of the present invention is realized as follows: A process flow for the verification and functional identification of lung cancer organoids, comprising the following steps: Step 1: Collect organoids; Step 2: Verify and functionally identify the organoids in Step 1; Among them, the verification and functional identification in Step 2 are: evaluating the developmental state; evaluating tumor characteristics; evaluating physiological functions; biological functions; identifying genetic characteristics; evaluating the immune microenvironment; evaluating drug sensitivity.

[0007] Further, the specific process of evaluating the developmental state in Step 2 is as follows: After sectioning the lung cancer organoid wax block, perform dewaxing with standard xylene, rehydration with gradient alcohol, staining with eosin and hematoxylin, and then mount the section to complete HE staining, and observe and acquire images under a microscope.

[0008] Further, the specific process of evaluating tumor characteristics in Step 2 is as follows: After dewaxing the organoid paraffin section with xylene and rehydrating it with gradient alcohol, perform chemical staining using an immunohistochemistry kit; Use the section without antibody as a negative control, observe and acquire images under a microscope after DAB color development; The tumor markers include Napsin A, BerEP4, CK7, MOC-31, TTF-1, CK5 / 6, and P40, and the consistency rate of the marker expression is 100%.

[0009] Further, the specific process of evaluating physiological functions in Step 2 is as follows: After dewaxing the organoid paraffin section with xylene and rehydrating it with gradient alcohol, perform chemical staining using an immunohistochemistry kit; Use the section without antibody as a negative control, observe and acquire images under a microscope after DAB color development; The proliferation markers of the organoids include Ki67 and PCNA, the apoptosis marker of the organoids is Cleaved-caspase3, and the epithelial-mesenchymal structure protein markers of the organoids include E-cadherin and Vimentin.

[0010] Further, the evaluation of biological functions in Step 2 includes identifying the secretory function of the organoids and identifying the pro-angiogenic ability; The specific process of identifying the secretory function of the organoids is as follows: Use Alcian blue staining, with the pH value of the Alcian blue being 2.5 or 1.0, for respectively identifying the secretion functions of acidic mucin and mucopolysaccharide protein; Deparaffinize the paraffin sections of the organoids in xylene, and then rehydrate them through gradient alcohol; Subsequently, when staining acidic mucin and mucopolysaccharide protein, add 100 μl of Alcian blue staining solution with a pH of 2.5 to each section, stain for 1 hour, wash the sections, then add 100 μl of nuclear fast red staining solution to each section, stain for 5 minutes, and wash with running water for 5 minutes; After the sections are dehydrated through gradient alcohol and cleared in xylene, seal them with neutral resin and observe and acquire images under a microscope; If staining the mucus substances secreted by cells, replace the Alcian blue staining solution with a pH of 1.0.

[0011] Furthermore, the specific process for identifying the angiogenesis-promoting ability is as follows: Thaw Matrigel divided into experimental and control groups at 4 °C, and then lay 100 μl per well in a 96-well plate, and place it at 37 °C for 30 minutes to solidify; Add 1 x 10 4 HUVEC cells containing basal culture medium to each well of the experimental and control groups. Among them, in the experimental group, culture the organoids in the supernatant for 48 hours and co-culture them with HUVEC cells, and in the control group, directly co-culture the organoids with HUVEC cells; Observe and acquire images under a microscope 1.5 h after culturing.

[0012] Furthermore, the specific process for identifying the genetic characteristics in step two is as follows: Use a DNA extraction kit to extract the DNA of the organoid section samples, and use the Trizol method to extract the total RNA; The extracted DNA and total RNA are detected for nucleic acid quality through Nanodrop and Qubit; The DNA is used for whole exome sequencing, and the total RNA is used for mRNA and lncRNA sequencing; The sequencing process mainly includes DNA or RNA library construction, sequencing data filtering and alignment analysis; The data analysis after DNA sequencing includes spectral analysis of the whole exome, COSMIC alignment analysis or heat map analysis; The data analysis after RNA sequencing includes differential expression genes of the transcriptome, expression quantity correlation analysis, overall distribution analysis of differential genes or immune microenvironment analysis; The higher the degree of genetic characteristic consistency of the organoids represents the higher the kinship between the organoids and the parental generation.

[0013] Furthermore, the specific process of evaluating the immune microenvironment in Step 2 is as follows: The consistency of the immune microenvironment is evaluated by RNA sequencing.

[0014] Furthermore, the specific process of evaluating drug sensitivity in Step 2 is as follows: The organoids are seeded in 96-well plates at a density of 50 per well, and the test drugs are serially diluted to set concentration gradients. Five parallel sub-wells are set up and cultured in a live cell imager for 48 hours, and the drug sensitivity is evaluated according to the change in the projected area of the organoids. Meanwhile, the sensitivity of the organoids to the drugs is compared with the efficacy of the clinical drugs used by the patients from whom the organoids are derived, and the patient efficacy is judged based on the results of computed tomography. After the drug sensitivity test, the organoids are washed with PBS and re-seeded in 96-well plates. The control group is a pure culture medium group without organoids. Detect using an LDH cytotoxicity detection kit. Measure the absorbance using a full-wavelength microplate reader at a wavelength of 490 nm, and perform dual-wavelength measurement using a wavelength of 600 nm as the reference wavelength. The absorbance measured for each group needs to subtract the absorbance of the pure culture medium group, and then perform LDH calculation.

[0015] Furthermore, the immune microenvironment of the organoids contains immune cell distribution characteristics consistent with those of the tumor tissue or cells from which they are derived.

[0016] Compared with the prior art, the present invention has the following advantages: The process for verifying and functionally identifying lung cancer organoids according to the present invention can ensure that the organoids are highly consistent with the parental tumors in terms of morphology, function, genetic characteristics, and immune microenvironment by establishing a comprehensive process for verifying and functionally identifying lung cancer organoids, thereby forming a standardized lung cancer organoid model. This process not only improves the reliability and accuracy of the organoid model, avoids the influence of unqualified samples on research results, but also promotes the application of organoid technology in drug research and development and personalized medicine, meets the needs of individualized drug use, reduces treatment side effects, and improves treatment effects. At the same time, this process enhances the understanding of tumor heterogeneity and microenvironment by comprehensively evaluating the physiological and pathological functions of organoids, solves the bottleneck problem in the field of organoid construction, promotes the development of precision lung cancer treatment, and provides strong support for improving the survival rate of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which form a part of the present invention, are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings: Figure 1 It is a schematic flow chart of the process for verifying and functionally identifying lung cancer organoids according to an embodiment of the present invention; Figure 2 It is for determining the basic morphology, developmental status and marker expression of organoids by HE staining and IHC staining in the process for verifying and functionally identifying lung cancer organoids according to an embodiment of the present invention; Figure 3 It is for evaluating the basic physiological functions of organoids by IHC staining in the process for verifying and functionally identifying lung cancer organoids according to an embodiment of the present invention; Figure 4 It is for evaluating the biological functions of organoids - secretion, angiogenesis promotion and phenotypic plasticity in the process for verifying and functionally identifying lung cancer organoids according to an embodiment of the present invention; Figure 5 It is for identifying the genetic characteristics of organoids at the DNA and RNA levels in the process for verifying and functionally identifying lung cancer organoids according to an embodiment of the present invention; Figure 6 It is for identifying the immune microenvironment of organoids in the process for verifying and functionally identifying lung cancer organoids according to an embodiment of the present invention; Figure 7 It is for identifying the drug sensitivity and cytotoxicity of organoids in the process for verifying and functionally identifying lung cancer organoids according to an embodiment of the present invention; Detailed implementation manners

[0018] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments may be combined with each other.

[0019] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by terms such as "upper", "lower", "inner", "back", etc. is based on the orientation or positional relationship shown in the accompanying drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and thus cannot be construed as a limitation to the present invention. In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance.

[0020] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments.

[0021] This embodiment relates to a process for verifying and functionally identifying lung cancer organoids, as Figure 1 shown, and includes the following steps: Step 1: Collect organoids; Step 2: Verify and functionally characterize the organoids obtained in Step 1; Among them, the verification and functional characterization in Step 2 include: evaluating the developmental status; evaluating the tumor characteristics; evaluating the physiological functions; biological functions; identifying the genetic characteristics; evaluating the immune microenvironment; evaluating the drug sensitivity.

[0022] The specific process for evaluating the developmental status in Step 2 is as follows: After sectioning the lung cancer organoid wax blocks, dewax with standard xylene, rehydrate with gradient ethanol, stain with eosin and hematoxylin, and then mount the slides to complete HE staining. Observe and acquire images under a microscope; as Figure 2 shown, morphological identification is performed by HE staining to determine that the organoids are tumor spheres formed by self-assembly and aggregation of cells. Complex organoids have hollow or solid structures, accompanied by epithelial and / or stromal structures.

[0023] The specific process for evaluating the tumor characteristics in Step 2 is as follows: After dewaxing the organoid paraffin sections with xylene and rehydrating with gradient ethanol, perform chemical staining using an immunohistochemistry kit; use the section without adding antibody as a negative control, observe and acquire images under a microscope after DAB color development; tumor markers include Napsin A, BerEP4, CK7, MOC-31, TTF-1, CK5 / 6, and P40, and the consistency rate of marker expression is 100%; as Figure 2 shown, through immunohistochemical staining, it is determined that the consistency rate of tumor marker expression between the lung cancer organoids and their source tissues or cells is 100%. These markers include Napsin A, BerEP4, CK7, MOC-31, TTF-1, CK5 / 6, and P40. Organoids with a consistency rate less than 100% are unqualified organoids.

[0024] The specific process for evaluating the physiological functions in Step 2 is as follows: After dewaxing the organoid paraffin sections with xylene and rehydrating with gradient ethanol, perform chemical staining using an immunohistochemistry kit; use the section without adding antibody as a negative control, observe and acquire images under a microscope after DAB color development; the proliferation markers of the organoids include Ki67 and PCNA, the apoptosis marker of the organoids is Cleaved-caspase3, and the epithelial-mesenchymal structure protein markers include E-cadherin and Vimentin; as Figure 3 shown, through immunohistochemical staining to identify the expression of proliferation, apoptosis, and epithelial-mesenchymal structure protein markers of the organoids, it is determined that the organoids have normal proliferation, apoptosis, and epithelial-mesenchymal structure transformation functions during physiological development.

[0025] The evaluation of biological functions in step 2 includes identifying the secretory function of the organoids and identifying the pro-angiogenic ability. The specific process for identifying the secretory function of the organoids is as follows: The pH values of Alcian blue staining are 2.5 or 1.0, which are used to identify the secretory functions of acidic mucin and mucopolysaccharide protein, respectively. The paraffin sections of the organoids are dewaxed in xylene and then rehydrated through gradient ethanol. Subsequently, when staining acidic mucin and mucopolysaccharide protein, 100 μl of Alcian blue staining solution with a pH of 2.5 is added dropwise to each section and stained for 1 hour. After washing the sections, 100 μl of nuclear fast red staining solution is added dropwise to each section and stained for 5 minutes, followed by washing with running water for 5 minutes. After the sections are dehydrated through gradient ethanol and cleared in xylene, they are mounted with neutral resin and observed and imaged under a microscope. When staining the mucus substances secreted by cells, the Alcian blue staining solution is replaced with a pH of 1.0.

[0026] The specific process for identifying the pro-angiogenic ability is as follows: Matrigel, which is divided into experimental and control groups, is thawed at 4 °C and then laid in 96-well plates at 100 μl per well, and placed at 37 °C for 30 minutes to solidify. 1 x 10 4 HUVEC cells containing basal culture medium are added to each well of the experimental and control groups. In the experimental group, the organoids are cultured in the supernatant for 48 hours and co-cultured with HUVEC cells, while in the control group, the organoids are directly co-cultured with HUVEC cells. Observation and imaging are carried out under a microscope 1.5 h after culturing.

[0027] As Figure 4 shown, it is identified that the organoids have secretory functions through Alcian blue staining, pro-angiogenic functions through tube formation assays, and phenotypic plasticity through physical induction of phenotypic transformation.

[0028] The specific process for identifying genetic characteristics in step 2 is as follows: DNA of the organoid section samples is extracted using a DNA extraction kit, and total RNA is extracted using the Trizol method. The extracted DNA and total RNA are detected for nucleic acid quality by Nanodrop and Qubit. The DNA is used for whole exome sequencing, and the total RNA is used for mRNA and lncRNA sequencing. The sequencing process mainly includes DNA or RNA library construction, sequencing data filtering, and alignment analysis. After DNA sequencing, data analysis includes spectral analysis of the whole exome, COSMIC alignment analysis, or heat map analysis. After RNA sequencing, data analysis includes differential expression genes in the transcriptome, expression quantity correlation analysis, overall distribution analysis of differential genes, or immune microenvironment analysis. The higher the degree of genetic characteristic consistency of the organoids, the higher the genetic relationship between the organoids and the parental generation.

[0029] As Figure 5 shown, the genetic characteristics of organoids were identified at two levels: DNA level and RNA level. At the DNA level, whole-exome sequencing was used to compare the gene mutation consistency between organoids and their source tissues or cells. At the RNA level, RNA sequencing was used to compare the consistency of mRNA and lncRNA expression. The higher the degree of genetic characteristic consistency, the higher the kinship between organoids and their parental generation. The consistency in this example can reach 95.59% - 97.21%.

[0030] The specific process of evaluating the immune microenvironment in Step 2 is as follows: The consistency of the immune microenvironment was evaluated by RNA sequencing; as Figure 6 shown, by RNA sequencing to evaluate the consistency of the proportion of immune cells in organoids and their source tumor tissues or cells, the immune microenvironment state of organoids can be fully reflected. The consistency in this example exceeds 90%.

[0031] The specific process of evaluating drug sensitivity in Step 2 is as follows: The organoids were inoculated into 96-well plates at a density of 50 per well, and the test drugs were serially diluted and concentration gradients were set. Five replicate wells were set up in parallel and cultured in a live cell imager for 48 hours. The drug sensitivity was evaluated based on the change in the projected area of the organoids. At the same time, the sensitivity of the organoids to the drugs was compared with the efficacy of the clinical drugs used by the patients from whom the organoids were derived. The patient efficacy was judged based on the results of computed tomography scans. After the drug sensitivity test, the organoids were washed with PBS and re-inoculated into 96-well plates. The control group was a pure culture medium group without organoids. The LDH cytotoxicity detection kit was used for detection. The absorbance was measured at a wavelength of 490 nm using a full-wavelength microplate reader, and a wavelength of 600 nm was used as the reference wavelength for dual-wavelength measurement. The absorbance measured for each group was subtracted by the absorbance of the pure culture medium group, and then the LDH was calculated.

[0032] As Figure 7 shown, through drug sensitivity testing and cytotoxicity testing, the purpose of verifying that the organoid model meets the requirements of individualized drug screening for patients was achieved. The organoids in this example can predict the clinical response of patients, and the cytotoxicity response and drug sensitivity are inversely proportional.

[0033] The immune microenvironment of organoids contains the same immune cell distribution characteristics as their source tumor tissues or cells.

[0034] This example was carried out as Figure 1The shown set of verification and identification processes can comprehensively evaluate the morphological characteristics, physiological and pathological functions of organoids to ensure that they possess various physiological and biological functions of lung cancer tumors, thereby forming a standardized lung cancer organoid model. Only organoids that take into account these characteristics and functions can maximally simulate or replace their original tumor cells for downstream industrial applications and scientific research.

[0035] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A process for validation and functional identification of lung cancer organoids, characterized in that: The following steps are involved: Step 1: Collect organoids; Step 2: verifying and functionally identifying the organoids obtained in step 1; Among them, the verification and functional identification in step two are: evaluating developmental status; evaluating tumor characteristics; evaluating physiological functions; biological functions; identifying genetic characteristics; evaluating immune microenvironment; and evaluating drug sensitivity.

2. The process flow for validation and functional identification of lung cancer organoids according to claim 1, characterized in that: The specific process of evaluating the developmental status in step 2 is as follows: After the lung cancer organoid wax blocks were sliced, they were sealed according to standard xylene dewaxing, gradient alcohol rehydration, eosin and hematoxylin staining, and HE staining was completed for observation and image acquisition under a microscope.

3. The process flow for validation and functional identification of lung cancer organoids according to claim 1, characterized in that: The specific process of evaluating tumor characteristics in step 2 is as follows: After the organoid paraffin sections were dewaxed with xylene and rehydrated with graded alcohol, chemical staining was performed using an immunohistochemistry kit; The sections without antibody were used as negative controls, and were observed and images were obtained under a microscope after DAB staining; The tumor markers include Napsin A, BerEP4, CK7, MOC-31, TTF-1, CK5 / 6 and P40, and the marker expression consistency rate is 100%.

4. The process flow for validation and functional identification of lung cancer organoids according to claim 1, characterized in that: The specific process of evaluating physiological function in step 2 is as follows: After the organoid paraffin sections were dewaxed with xylene and rehydrated with graded alcohol, chemical staining was performed using an immunohistochemistry kit; The sections without antibody were used as negative controls, and were observed and images were obtained under a microscope after DAB staining; The proliferation markers of the organoids include Ki67 and PCNA, the apoptosis marker of the organoids is Cleaved-caspase3, and the epithelial-mesenchymal structural protein markers of the organoids include E-cadherin and Vimentin.

5. The process flow for validation and functional identification of lung cancer organoids according to claim 1, characterized in that: The evaluation of biological functions in step 2 includes identifying the secretory function of the organoid and identifying the angiogenesis-promoting ability; The specific process of identifying the secretory function of the organoid is as follows: Using Alcian blue staining, wherein the pH value of Alcian blue is 2.5 or 1.0, to identify the secretory function of acidic mucin and mucopolysaccharide protein, respectively; Organoid paraffin sections were dewaxed in xylene and rehydrated in graded alcohols; When staining acidic mucin and mucopolysaccharide proteins, 100 μl of Alcian blue staining solution with a pH of 2.5 was then added to each section for 1 hour, and after washing the sections, 100 μl of nuclear fast red staining solution was added to each section for 5 minutes, followed by washing with running water for 5 minutes; The sections were dehydrated with graded alcohol, transparentized with xylene, and sealed with neutral resin for observation and image acquisition under a microscope; If staining mucus secreted by cells, change the Alcian blue staining solution to pH 1.

0.

6. The process flow for validation and functional identification of lung cancer organoids according to claim 5, characterized in that: The specific process of identifying the angiogenesis-promoting ability is as follows: The Matrigel divided into the experimental group and the control group was thawed at 4°C and spread in 96-well plates at 100 μl per well, and placed at 37°C for 30 minutes to solidify; 1 x 10 HUVEC cells containing basal culture medium were added to each well of the experimental group and the control group. 4 In the experimental group, the organoids were cultured in the supernatant for 48 h and co-cultured with HUVEC cells, while in the control group, the organoids were directly co-cultured with HUVEC cells; Observe and acquire images under a microscope 1.5 hours after incubation.

7. The process flow for validation and functional identification of lung cancer organoids according to claim 1, characterized in that: The specific process of identifying genetic characteristics in step 2 is as follows: DNA from organoid slice samples was extracted using a DNA extraction kit, and total RNA was extracted using the Trizol method; The extracted DNA and total RNA were tested for nucleic acid quality using Nanodrop and Qubit; DNA was used for whole exome sequencing, and total RNA was used for mRNA and lncRNA sequencing; The sequencing process mainly includes DNA or RNA library construction, sequencing data filtering and comparison analysis; Post-DNA sequencing data analysis includes whole-exome spectrum analysis, COSMIC alignment analysis, or heat map analysis; Data analysis after RNA sequencing includes differentially expressed genes of the transcriptome, expression correlation analysis, overall distribution analysis of differentially expressed genes, or immune microenvironment analysis; The higher the degree of consistency of the genetic characteristics of the organoids, the higher the affinity between the organoids and their parents.

8. The process flow for validation and functional identification of lung cancer organoids according to claim 1, characterized in that: The specific process of evaluating the immune microenvironment in step 2 is as follows: The identity of the immune microenvironment was assessed by RNA sequencing.

9. The process flow for validation and functional identification of lung cancer organoids according to claim 1, characterized in that: The specific process of evaluating drug sensitivity in step 2 is as follows: The organoids were seeded in a 96-well plate at a density of 50 per well, and the drugs to be tested were diluted in multiples and a concentration gradient was set; Five replicate wells were set up in parallel and cultured for 48 hours in a live cell imager to evaluate drug sensitivity based on changes in the projected area of ​​the organoids; At the same time, the sensitivity of organoids to drugs was compared with the efficacy of clinical drugs used by patients from which the organoids were derived, and the efficacy of patients was determined based on the results of electronic computed tomography scans; After the drug sensitivity test, the organoids were washed with PBS and then re-seeded in a 96-well plate. The control group was a pure culture medium group without organoids; Detected using LDH cytotoxicity detection kit; The absorbance was measured at 490 nm using a full-wavelength microplate reader, and dual-wavelength determination was performed using 600 nm as the reference wavelength; The absorbance of each group measured must be subtracted from the absorbance of the pure culture solution group before LDH calculation.

10. The process flow for validation and functional identification of lung cancer organoids according to claim 8, characterized in that: The immune microenvironment of the organoid contains immune cell distribution characteristics consistent with the tumor tissue or cells from which it is derived.

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