A novel method for constructing a lung cancer brain metastasis model

Abstract

This invention relates to the field of biotechnology, and more particularly to a novel method for constructing a lung cancer brain metastasis model. The method includes: inducing stem cell culture to obtain embryoid organoids; differentiating and culturing the embryoid organoids to obtain ventral midbrain organoids; mincing and digesting small cell lung cancer (SCLC) tissue blocks to obtain SCLC cells; culturing these cells in suspension to obtain SCLC organoids; and co-culturing the ventral midbrain organoids and SCLC organoids in the same culture container to obtain the novel lung cancer brain metastasis model. Compared to the lower success rate of human tumor tissue xenotransplantation, the novel lung cancer brain metastasis model provided by this invention has higher fusion efficiency and batch stability, and can also reduce interference from other xenogeneic animal components. Furthermore, the organoid fusion model established using this invention is easier to observe, administer drugs to, and sample.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a novel method for constructing a lung cancer brain metastasis model. Background Technology

[0002] Small cell lung cancer (SCLC) is a highly malignant neuroendocrine-derived lung cancer, accounting for 15%-20% of all lung cancer cases. Its cancer cells are small, rapidly dividing, with a doubling time of only about 33 days, and can metastasize extensively even in the early stages of the cancer. Clinicians need to develop personalized treatment plans for brain metastases from small cell lung cancer based on the specific circumstances of each patient, and rely heavily on disease models of brain metastases from small cell lung cancer. Currently, biological experimental models for studying brain metastases from small cell lung cancer mainly include animal models, organoids, and microfluidic technology systems. Animal models are divided into genetically engineered mouse models (systematic knockout of the Rb / p53 dual genes) and SCLC cell line (or organoid) transplantation models. Microfluidic chips simulate the brain microenvironment, enabling the co-culture of tumor cells and astrocytes. Furthermore, it has been found that SCLC cells can form functional synapses with neurons and promote tumor growth through neurotransmitter signals, revealing the regulatory mechanism of the nervous system on SCLC. However, there are currently no publicly reported models that explicitly mention the direct fusion of small cell lung cancer organoids and brain organoids, but research progress in related fields has provided a technical foundation and research ideas for this direction.

[0003] Existing methods all have significant limitations. SCLC cancerous tissue isolated from patients, after being processed into monolayers or spherical cell lines, requires considerable time to expand and establish a complete system, and it is difficult to maintain the diversity of the original tumor cell population and the expression of key driver genes. Currently, researchers believe that human tumor tissue xenograft models can better preserve the characteristics of the original tumor; however, the transplantation efficiency of these models is highly unstable, and the significant difference between the host environment and the human body leads to substantial differences in subsequent drug delivery, response, and metabolism. Although studies have reported on the fusion of SCLC organoids with astrocytes to generate functional synapses, astrocytes lack the heterogeneity and biochemical diversity of brain organoid cell populations, meaning they cannot better simulate the real microenvironment of SCLC brain metastases in vivo. Currently, there are no publicly reported models explicitly mentioning the direct fusion of small cell lung cancer organoids and brain organoids. Therefore, developing new in vitro models that can maintain the proliferation of small cell lung cancer while ensuring cellular heterogeneity will help advance research related to personalized treatment for SCLC. Summary of the Invention

[0004] To address the technical problems existing in the prior art, the present invention aims to provide a novel method for constructing a lung cancer brain metastasis model to solve the aforementioned technical problems. The main technical problem solved by the present invention is to develop a novel in vitro model that can maintain the proliferation of small cell lung cancer while ensuring heterogeneity among cells.

[0005] To achieve the above-mentioned objectives, the present invention adopts the following technical solution.

[0006] According to a first aspect of the present invention, the present invention provides a novel method for constructing a lung cancer brain metastasis model (a method for preparing a chimeric culture model of ventral midbrain organoids and SCLC organoids), comprising the following steps:

[0007] (1) Stem cells were induced to culture to obtain embryonic organoids, and the embryonic organoids were differentiated and cultured to obtain ventral midbrain organoids;

[0008] (2) The SCLC tissue blocks were cut into pieces, digested, and SCLC cells were obtained. The cells were then cultured in suspension to obtain small cell lung cancer organoids.

[0009] (3) The ventral midbrain organoids and small cell lung cancer organoids were placed in the same culture container and co-cultured to obtain the novel lung cancer brain metastasis model.

[0010] In some embodiments, the stem cells in step (1) are human induced pluripotent stem cells (IPSCs).

[0011] In some embodiments, step (1) of inducing stem cell culture includes: transferring 5,000 to 15,000 human induced pluripotent stem cells (IPSCs) digested into a single morphology to an ultra-low adsorption 96-well plate for induction culture.

[0012] In some embodiments, the induction culture in step (1) includes the following steps: placing stem cells in a neural induction medium containing a smoothing agonist, CHIR99021, and Y-27632 for a first induction culture treatment, and then in a medium containing Noggin, SB431542 (a Src family kinase inhibitor), and CHIR99021 (chemical formula: The embryoid organoids were obtained by performing a second induction culture treatment in a neural induction medium containing smoothing agonist and FGF-8, followed by a third induction culture treatment in a neural induction medium containing smoothing agonist and FGF-8.

[0013] In some embodiments, the preparation of the neural induction culture medium includes:

[0014] Add N2 supplement, L-alanyl-L-glutamine solution, MEM-Eagle medium containing non-essential amino acids, heparin solution, and penicillin-streptomycin solution to DMEM / F12 medium, mix well, and obtain the neural induction medium.

[0015] In some embodiments, the N2 supplement in the neural induction medium comprises 0.5% to 2% by volume, the L-alanyl-L-glutamine solution comprises 0.5% to 2% by volume, the MEM-Eagle medium containing non-essential amino acids comprises 0.5% to 2% by volume, and the penicillin-streptomycin solution comprises 0.5% to 2% by volume; the volume ratio of the heparin solution to the neural induction medium is 1:500 to 1:2000.

[0016] In some embodiments, the concentration of the L-alanyl-L-glutamine solution is 200 mM; the concentration of the non-essential amino acids in the MEM-Eagle medium containing non-essential amino acids is 10 mM; the concentration of the heparin solution is 1 mg / mL; and the concentration of penicillin in the penicillin-streptomycin dual antibody solution is 10000 U / mL and the concentration of streptomycin is 10 mg / mL.

[0017] In some embodiments, the induction culture in step (1) includes: placing stem cells in a neural induction medium containing 5-15 µM of a smoothing agonist, 0.5-2 µM of CHIR99021, and 0.5-1 µM of Y-27632 for a first induction culture treatment of 1.5-2.5 days; then performing a second induction culture treatment in a neural induction medium containing 100-300 ng / ml of Noggin, 5-15 µM of SB431542, 0.5-2 µM of CHIR99021, 200-500 nM of a smoothing agonist, and 20-200 ng / ml of FGF-8 for a second induction culture treatment of 3.5-4.5 days; followed by induction culture in a neural induction medium containing 200-500 nM of a smoothing agonist and 20-200 ng / ml of FGF-8. The embryoid organoids were obtained by performing a third induction culture treatment with ng / ml FGF-8 neural induction medium for 1.5-2.5 days.

[0018] In some embodiments, the differentiation culture in step (1) includes the following steps: placing the embryoid organoid in Improved-A differentiation medium containing liquid matrix gel, smoothing agonist and FGF-8 for a first static culture, then in Improved-A differentiation medium for a second static culture, then in Improved+A differentiation medium for a third static culture, and finally in a mixture of Improved+A differentiation medium and Brainphys differentiation medium for a fourth static culture to obtain the ventral midbrain organoid.

[0019] In some embodiments, the differentiation culture in step (1) includes the following steps: placing the embryoid organoid in a solution containing a liquid matrix gel at a volume percentage concentration of 1% to 3%, a smoothing agonist at a concentration of 200 to 500 nM, and a solution containing 20 to 200 nM. The culture was first statically cultured in Improved-A differentiation medium containing ng / ml of FGF-8 for 2.5-3.5 days. Then, a second static culture was performed in Improved-A differentiation medium for 3.5-4.5 days. Next, a third static culture was performed in Improved+A differentiation medium for 3.5-4.5 days. Finally, a fourth static culture was performed in a mixture of Improved+A and Brainphys differentiation medium. At the beginning of the fourth static culture, the volume percentage of Brainphys differentiation medium in the mixture was 7-8%. The medium was changed every 2-4 days, increasing the volume percentage of Brainphys differentiation medium by 7-8% each time, until the medium was replaced with 100% Brainphys differentiation medium. After replacing with 100% Brainphys differentiation medium, mature ventral midbrain organoids were obtained.

[0020] In some embodiments, step (2) of cutting and digesting SCLC tissue blocks to obtain SCLC cells includes: removing SCLC tissue blocks from the patient, soaking them in physiological saline, and cutting them thoroughly with surgical scissors until more than 90% of the tissue fragments have an average diameter of less than 1 cm. After centrifugation, the tissue fragments are collected, the supernatant is removed, and the fragments are resuspended in digestion solution I. The mixture is then placed in a 37°C water bath for 20 to 40 minutes, during which it is vortexed multiple times. Under a microscope, the small cell lung cancer tissue is observed to see if it has been fully digested into single cells or small cell clusters of less than 7 cells. Then, DMEM medium containing 10 wt% fetal bovine serum is added to terminate the digestion. The cell pellet is collected by centrifugation to obtain SCLC cells.

[0021] In some embodiments, the process of obtaining SCLC cells in step (2) and culturing them in suspension includes: resuspending the cell pellet in small cell lung cancer culture medium, maintaining suspension culture, and allowing small cell lung cancer organoids to differentiate and mature after 2 weeks.

[0022] In some embodiments, the suspension culture in step (2) includes the following steps: placing SCLC cells in small cell lung cancer tissue culture medium (Accurate, M123) for culture, wherein the initial cell density of the SCLC cells in the small cell lung cancer tissue culture medium (labeled as SCLC medium) is 30 to 1,000,000 cells / mL, and the culture time is 13 to 15 days.

[0023] In some embodiments, the volume ratio of the ventral midbrain organoids and the small cell lung cancer organoids in step (3) is 2:1 to 10:1; the co-culture includes the following steps:

[0024] Midbrain ventral organoids (preferably midbrain ventral organoids differentiated from IPSC for more than 60 days) and small cell lung cancer organoids were placed in the same culture container (a 24-well plate with ultra-low adsorption is optional), and Brainphys differentiation medium and an equal volume of SCLC medium were added (the amount of Brainphys differentiation medium and SCLC medium added were both 300~500 μL). Then, they were cultured on a shaker at a speed of 50-200 rpm for 10-15 days.

[0025] In some implementations, the culture medium is changed every three days during the co-culture process.

[0026] In some implementations, during co-culture, step (3) can be photographed every 1 to 2 days to record the growth of the novel lung cancer brain metastasis model. After 10 to 15 days of co-culture, some fused organoids (the novel lung cancer brain metastasis model) can be taken out as samples and then made into frozen sections with a thickness of 8 to 14 micrometers. Immunohistochemistry is used to identify the relevant markers of the ventral midbrain organoids and small cell lung cancer.

[0027] In some implementations, in step (1), during the 7-10 days of induction culture, embryonic organoids can be transferred to 6 cm Petri dishes or ultra-low adsorption 6-well plates that have undergone low adsorption treatment in advance using a Pasteur pipette. The number of embryonic organoids in the 6 cm Petri dish is kept to be 5-14, and the number of organoids in the ultra-low adsorption 6-well plate is not more than 4. The differentiation efficiency of mature ventral midbrain organoids cultured in smaller culture containers will be higher.

[0028] According to a second aspect of the present invention, the present invention provides a novel lung cancer brain metastasis model obtained by the above-described method for constructing a novel lung cancer brain metastasis model.

[0029] According to a third aspect of the present invention, the present invention provides the application of a novel lung cancer brain metastasis model in SCLC drug development.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] (1) Compared with monolayer adherent culture and suspension spheroid culture of small cell lung cancer, the method provided in this embodiment of the invention can maintain the 3D morphology of SCLC tumor organoids while maintaining the effective proliferation of SCLC tumors.

[0032] (2) Compared with the lower success rate of human tumor tissue xenotransplantation technology, the novel lung cancer brain metastasis model construction method provided in this embodiment of the invention has higher fusion efficiency and batch stability, and can also reduce interference from other xenogeneic animal components. In addition, the organoid fusion model established using this invention technology is easier to observe, administer drugs, and sample.

[0033] (3) Compared with the fusion model of SCLC cell organoids and glial cells, the novel lung cancer brain metastasis model (a model of fusion of brain and lung organoids) provided in this embodiment of the invention has cell populations and biochemical characteristics that are closer to the real scenario of SCLC brain metastasis in the human body. Attached Figure Description

[0034] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 This is a bright-field image of the fusion culture of ventral brain organoids and SCLC organoids in an embodiment of the present invention after 15 days;

[0036] Figure 2 These are live cell fluorescence staining images of brain ventral organoids and SCLC organoids fused together after 15 days in an embodiment of the present invention.

[0037] Note: The cellular organoid clumps attached to the edge and not labeled with green fluorescence are SCLC organoids, while the approximately circular outline region labeled with GFP fluorescence belongs to the ventral midbrain organoid (VM).

[0038] Figure 3 Microscopic images of the ventral midbrain organoid fused with the SCLC organoid 15 days after fusion. Detailed Implementation

[0039] To better understand the technical solution of the present invention, the embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0040] It should be understood that the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0041] The main reagents and consumables used in the following examples are shown in Table 1 below.

[0042] Table 1

[0043]

[0044]

[0045] Unless otherwise specified, all cultures in the following examples were conducted at a temperature of 37°C and a 5% CO2 atmosphere.

[0046] The culture media used in the following examples are described below.

[0047] The formulation of the neural induction medium is as follows: Using DMEM / F12 medium as the stock solution, add 0.5%–2% N2 supplement, 0.5%–2% L-alanyl-L-glutamine solution (200 mM), 0.5%–2% MEM-Eagle medium containing non-essential amino acids, 1:500–1:2000 heparin solution (1 mg / ml), and 0.5%–2% penicillin-streptomycin antibiotics. % indicates volume percentage.

[0048] The improved-A differentiation medium formulation is as follows: A stock solution is a 1:1 mixture of DMEM / F12 and Neurobasal media, supplemented with 0.2%–1% N2 supplement, 1%–4% B27-A supplement, 5–12 mg / ml insulin, 0.5%–2% L-alanyl-L-glutamine solution (200 mM), 0.2%–1% MEM-Eagle medium containing non-essential amino acids, and 0.5%–2% penicillin-streptomycin antibiotics. % indicates volume percentage.

[0049] The formulation of the Improved+A differentiation medium is as follows: A stock solution is prepared by mixing DMEM / F12 and Neurobasal medium in a 1:1 ratio, supplemented with 0.1%–1% MEM-Eagle medium containing non-essential amino acids, 1%–4% B27+A supplement, 5–12 mg / ml insulin, 0.5%–2% L-alanyl-L-glutamine solution (200 mM), 0.5%–2% penicillin-streptomycin antibiotics, 0.5%–2% ascorbic acid solution (40 mM), and 0.5–2 g / L sodium bicarbonate. % indicates volume percentage.

[0050] The formulation of BrainPhys differentiation medium is as follows: using BrainPhys Neuronal Medium as the stock solution, add 1%–4% B27+A supplement, 0.5%–2% N2 supplement, 1–3 ml CD Lipid Concentrate (50 ml medium system), 0.5%–2% penicillin-streptomycin antibiotics, 1:100–1:200 glucose solution (20%), 10–30 ng / ml brain-derived neurotrophic factor, 10–30 ng / ml glial cell-derived neurotrophic factor, and 0.5–2 mM bis(2-ethylhexyl) cyclic adenosine monophosphate (bcAMP). % indicates volume percentage.

[0051] Example 1

[0052] A novel method for constructing a lung cancer brain metastasis model includes the following steps:

[0053] (1) Obtaining cell suspension: IPSCs were seeded in 6-well plates and cultured in each well with 2 ml of mTeSR medium (Stemcell, cat. no. 85850) until 85%–90% confluence. The old medium was then removed, and 1 ml of DPBS buffer (Dulbecco's phosphate buffer, Thermo Fisher, cat. no. 14190367) was added to wash the bottom of the plate. Accutase cell digestion solution (STEM CELL, cat. no. 07920) was added to each well and incubated at 37°C for 5 minutes. The digestion reaction was terminated by adding 3 ml of mTeSR medium. The cells were then transferred to 15 ml centrifuge tubes and centrifuged at 1400 rpm for 5 minutes. The cell pellet was collected. The supernatant was removed, and the cells were resuspended in 1 ml of mTeSR medium for cell counting. After counting, the IPSC suspension was centrifuged at 1400 rpm for 5 minutes, the supernatant was discarded and the cell pellet was obtained. The cell pellet was resuspended in neural induction medium containing 10 µM smoothing agonist (Merck, cat. no. US1566660), 1 µM CHIR99021 (Tocris, cat. no. 4423), and 1 μM Y-27632 (Wako, Cat. no. 030-24021) to obtain a cell suspension.

[0054] Induction culture treatment: Cell suspension was transferred to 9000 cells / well in ultra-low adsorption 96-well plates (CORNING, cat. no. 3474), with the total volume of medium in each well controlled at 150 μL. Induction culture treatment was carried out at 37°C. Then, on the 2nd and 4th days after the start of induction culture treatment, the medium was replaced with fresh neural induction medium containing 250 ng / ml Noggin (R&D Systems, cat. no. 6057), 10 µM SB431542 (STEMCELL TECHNOLOGIES, cat. no. 72232), 1 µM CHIR99021, 300 nM smoothing agonist, and 100 ng / ml human FGF-8 recombinant protein, respectively. On the 6th day after the start of induction culture treatment, the medium was replaced with neural induction medium containing 300 nM smoothing agonist and 100 ng / ml FGF-8. After 2 days of culture, embryoid organoids were obtained. Eight days before the start of induction culture, prepare ultra-low adsorption petri dishes. Add 5 ml of anti-adhesion washing solution to a newly opened 10 cm cell culture dish, shake well to spread it evenly to cover the entire dish, and store it in a 37°C incubator.

[0055] Differentiation induction treatment: On day 8 after the start of induction culture, anti-adhesion washing buffer was aspirated from the 10 cm cell culture dish, and 4 ml of PBS was added three times to wash the surface of the dish. After the surface of the dish was dried with a pipette tip, 15 embryoid organoids were transferred to each 10 cm cell culture dish with low adsorption using a Pasteur pipette. 12 ml of Improved-A differentiation medium containing 2% liquid matrix gel, 300 nM smoothing agonist and 100 ng / ml human FGF-8 recombinant protein (R&D Systems, cat. no. 5057-FF) was added, and the dish was incubated statically for 3 days. On day 12, the medium was replaced with 12 ml of Improved-A differentiation medium without other factors.

[0056] From day 16, replace the culture medium with 12 ml of Improved+A differentiation medium, and change the medium every 3 days. From day 20, maintain the total volume of the culture medium at 30 ml, and gradually add 7% or 8% Brainphys differentiation medium (% represents volume percentage) to the culture medium each time the medium is changed, and change the medium every 3 days. From day 60, replace the culture medium with 100% Brainphys differentiation medium, and keep the organoids cultured in 100% Brainphys differentiation medium until they differentiate into mature ventral midbrain organoids. Change the medium every 3 days. Do not freeze.

[0057] (4) Small cell lung cancer tissue was excised from the patient and immersed in a centrifuge tube containing 50 ml of physiological saline. Some of the saline was discarded, but the saline level was kept 2 cm above the tissue. Using surgical scissors, the tissue was thoroughly cut into small pieces (the average diameter of the fragments was less than 1 cm). The centrifuge tube was filled with physiological saline and inverted several times to allow the tissue fragments to detach from the centrifuge tube wall. The tube was centrifuged at 300 x g for 5 minutes to collect the cell pellet. The supernatant was removed and the pellet was resuspended in 10 ml of digestion solution I (Gibco, cat. no. 12605028). The pellet was then incubated in a 37°C water bath for 20 minutes, during which time the centrifuge tube was inverted 10 times every 5 minutes. The pellet was vortexed and observed under a microscope to see if the SCLC had been adequately digested into single cells or cell clusters of fewer than 7 cells. Digestion was terminated by adding the same volume of DMEM medium (Gibco, cat. no. C11995500BT) containing 10% fetal bovine serum (Gibco, cat. no. 30044333) as digestion solution I. The cell pellet was collected by centrifugation at 300 ×g and resuspended in small cell lung cancer tissue culture medium (Accurate, cat. no. M123). The cells were then transferred to low-adsorption Petri dishes for suspension culture, maintaining a cell density of 500,000 cells / mL. The culture medium was changed every 3 days. After 2 weeks, the cells differentiated into relatively mature SCLC organoids.

[0058] (5) Using a 3 mL Pasteur pipette, 20 ventral midbrain organoids differentiated from IPSCs for more than 60 days were taken from a low-adsorption Petri dish and transferred to a 24-well plate with ultra-low adsorption (1 organoid / well). Then, SCLC organoids were taken and added to wells containing one or two ventral midbrain organoids. The volume of the ventral midbrain organoids in each well of the 24-well plate was 2:1 with the total volume of the SCLC organoids. 350 μL of Brainphys differentiation medium was added to each well, along with an equal volume of SCLC medium. The plates were then centrifuged at 50 rpm on a horizontal shaker and cultured continuously, with the medium changed every three days. After 15 days of co-culture, the results of bright-field observation under a 10x microscope are as follows: Figure 1 The results of live-cell fluorescence observation under a 4x microscope are shown below. Figure 2 As shown ( Figure 2 The green fluorescent markers in the image are organoids from the ventral midbrain region. Figure 1 and Figure 2 The results showed that the ventral midbrain organoids and the SCLC organoids had fused.

[0059] The fused organoids (those co-cultured for 15 days) were extracted, frozen sections were prepared, and immunohistochemistry was performed according to the instructions of the HRP / DABIHC Detection kit (Abcam, ab236466) to identify relevant markers of the fused organoids of the midbrain ventral organoid and SCLC organoid, including two markers, GFAP and NKX2.1.

[0060] Immunohistochemical results also showed that both generated significant GFAP neurons. + Synaptic connections, namely GFAP expression, encompass the periphery of both organoids ( Figure 3 As shown in Part A (field of view under 10x magnification), and multiple individual SCLC organoids (primarily composed of NKX2.1). + The cells (composed of cells) attach to multiple sites on the surface of the ventral organoids of the midbrain, but do not invade the interior of the brain organoids. Figure 3 As shown in Part B, the field of view under a 4x scope. Figure 3 The cellular region enclosed by the yellow dashed circle in Part B belongs to the SCLC organoid.

[0061] The above descriptions are merely some embodiments of the present invention. Those skilled in the art can make various modifications and improvements without departing from the inventive concept of the present invention, and these all fall within the scope of protection of the present invention.

Claims

1. A method for constructing a lung cancer brain metastasis model, characterized in that, Includes the following steps: (1) Stem cells were induced to culture to obtain embryonic organoids, and the embryonic organoids were differentiated and cultured to obtain ventral midbrain organoids; (2) The small cell lung cancer tissue block was cut into pieces, digested, and small cell lung cancer cells were obtained. The cells were then cultured in suspension to obtain small cell lung cancer organoids. (3) The ventral midbrain organoids and small cell lung cancer organoids were placed in the same culture container and co-cultured to obtain the lung cancer brain metastasis model; The stem cells mentioned in step (1) are human induced pluripotent stem cells; Step (1) of the induction culture includes: placing stem cells in a neural induction medium containing 5-15 µM of smoothing agonist, 0.5-2 µM of CHIR99021, and 0.5-1 µM of Y-27632 for a first induction culture treatment of 1.5-2.5 days; then performing a second induction culture treatment in a neural induction medium containing 100-300 ng / ml of Noggin, 5-15 µM of SB431542, 0.5-2 µM of CHIR99021, 200-500 nM of smoothing agonist, and 20-200 ng / ml of FGF-8 for a second induction culture treatment of 3.5-4.5 days; followed by induction culture in a neural induction medium containing 200-500 nM of smoothing agonist and 20-200 ng / ml of FGF-8. The embryoid organoids were obtained by performing a third induction culture treatment with ng / ml FGF-8 neural induction medium for 1.5-2.5 days. The differentiation culture described in step (1) includes the following steps: placing embryoid organoids in a solution containing 2% liquid matrix gel (volume percentage concentration), 200-500 nM smoothing agonist, and 20-200 nM smoothing agonist. The culture was first statically incubated for 2.5-3.5 days in Improved-A differentiation medium at ng / ml, followed by a second static incubation for 3.5-4.5 days. Then, a third static incubation was performed in Improved+A differentiation medium for another 3.5-4.5 days. Finally, a fourth static incubation was performed in a mixture of Improved+A and Brainphys differentiation medium. At the start of the fourth static incubation, the volume percentage of Brainphys differentiation medium in the mixture was 7-8%. The medium was changed every 2-4 days, increasing the volume percentage of Brainphys differentiation medium by 7-8% each time, until the medium was completely replaced with 100% Brainphys differentiation medium. After differentiation into Brainphys culture medium, ventral midbrain organoids can be obtained; the smooth agonist was purchased from Merck, cat. no. US1566660. The suspension culture described in step (2) includes the following steps: Small cell lung cancer cells were cultured in small cell lung cancer tissue culture medium, with an initial cell density of 300,000 to 1,000,000 cells / mL and a culture time of 13 to 15 days. The volume ratio of the ventral midbrain organoids and the small cell lung cancer organoids in step (3) is 2:1-10:1; the co-culture in step (3) includes the following steps: The ventral midbrain organoids and small cell lung cancer organoids were placed in the same culture container, and Brainphys differentiation medium and an equal volume of small cell lung cancer medium were added. They were then cultured on a shaker at a speed of 50-200 rpm for 10-15 days. Improved-A differentiation medium consisted of a stock solution of a 1:1 mixture of DMEM / F12 and Neurobasal medium, supplemented with 0.2%–1% N2 supplement, 1%–4% B27-A supplement, 5–12 mg / ml insulin, 0.5%–2% 200 mM L-alanyl-L-glutamine solution, 0.2%–1% MEM-Eagle medium containing non-essential amino acids, and 0.5%–2% penicillin-streptomycin antibiotics. % indicates volume percentage. Improved+A differentiation medium consisted of a stock solution of a 1:1 mixture of DMEM / F12 and Neurobasal medium, supplemented with 0.1%–1% MEM-Eagle medium containing non-essential amino acids, 1%–4% B27+A supplement, 5–12 mg / ml insulin, 0.5%–2% 200 mM L-alanyl-L-glutamine solution, 0.5%–2% penicillin-streptomycin antibiotics, 0.5%–2% 40 mM antiascorbic acid solution, and 0.5–2 g / L sodium bicarbonate. % indicates volume percentage. The formulation of BrainPhys differentiation medium is as follows: using BrainPhys Neuronal Medium as the stock solution, add 1%~4% B27+A supplement, 0.5%~2% N2 supplement, 1~3 ml CD Lipid Concentrate, 0.5%~2% penicillin-streptomycin antibiotics, 1:100~1:200 20% glucose solution, 10~30 ng / ml brain-derived neurotrophic factor, 10~30 ng / ml glial cell-derived neurotrophic factor, and 0.5~2mM bis(butyryl) cyclic adenosine monophosphate sodium salt. % indicates volume percentage.

2. The method for constructing a lung cancer brain metastasis model according to claim 1, characterized in that, The preparation of the neural induction culture medium includes: Add N2 supplement, L-alanyl-L-glutamine solution, MEM-Eagle medium containing non-essential amino acids, heparin solution, and penicillin-streptomycin solution to DMEM / F12 medium, mix well, and obtain the neural induction medium.

3. The method for constructing a lung cancer brain metastasis model according to claim 2, characterized in that, In the neural induction medium, the volume percentage of N2 supplement is 0.5% to 2%, the volume percentage of L-alanyl-L-glutamine solution is 0.5% to 2%, the volume percentage of MEM-Eagle medium containing non-essential amino acids is 0.5% to 2%, and the volume percentage of penicillin-streptomycin solution is 0.5% to 2%; the volume ratio of heparin solution to neural induction medium is 1:500 to 1:2000.

4. The method for constructing a lung cancer brain metastasis model according to claim 3, characterized in that, The concentration of the L-alanyl-L-glutamine solution is 200 mM; the concentration of non-essential amino acids in the MEM-Eagle medium containing non-essential amino acids is 10 mM; the concentration of the heparin solution is 1 mg / mL; and the concentration of penicillin in the penicillin-streptomycin dual antibody solution is 10000 U / mL and the concentration of streptomycin is 10 mg / mL.

5. A lung cancer brain metastasis model obtained by the method for constructing a lung cancer brain metastasis model according to any one of claims 1-4.

6. The application of the lung cancer brain metastasis model as described in claim 5 in the development of drugs for small cell lung cancer.

Images