Small cell lung cancer organoid culture medium and culture method

Abstract

The invention provides a special culture medium for small cell lung cancer organoid and application of the special culture medium. The culture medium for the small cell lung cancer organoid comprises a basic culture medium, an antimicrobial agent, a culture medium supplement, cell factors and an optional buffering agent. The culture medium can be used for continuously culturing small-cell lung cancer tumor organs in vitro, can well maintain the invasion characteristics of small-cell lung cancer tumors, realizes in-vitro research on the specificity and drug resistance mechanism of small-cell lung cancer tumors, and has important clinical application value for mining potential targeted therapy targets of small-cell lung cancer.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, specifically to a culture medium and culture method suitable for culturing organoids from small cell lung cancer with multiple gene deletions. Background Technology

[0002] Lung cancer is a primary malignant tumor of the lung originating from the bronchial mucosa or glands. Based on pathological characteristics, it can be divided into two main categories: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Small cell lung cancer accounts for approximately 15% of all lung cancer cases. It is a malignant tumor with abnormal proliferation and invasiveness, characterized by gene inactivation such as p53 and Rb1, rapid tumor metastasis, and resistance to radiotherapy and chemotherapy. Due to the lack of effective treatments, SCLC patients have a poor clinical prognosis, with a 5-year survival rate of approximately 8-10% and a median overall survival of 10-12 months.

[0003] Currently, because most SCLC patients have already metastasized upon diagnosis and rarely undergo surgical resection, the possibility of obtaining in vitro clinical samples of SCLC and conducting in-depth research on SCLC tumor cells is limited. While SCLC transgenic mice and related cell lines are widely used in vitro to study the regulatory mechanisms of SCLC malignant proliferation and metastasis, these models still lack certain guiding significance. This is mainly because the metastatic characteristics of SCLC transgenic mouse models cannot simulate the real situation of SCLC patients, and SCLC cell lines cannot reflect the specific microenvironment of SCLC tumor cells. Tumor organoids, on the other hand, possess a better three-dimensional structure and can better maintain tumor characteristics and the tumor microenvironment, providing a powerful tool for in vitro research on SCLC tumor heterogeneity, malignant invasion, and metastasis.

[0004] Organoids are 3D cell cultures created in vitro using embryonic stem cells, pluripotent stem cells, and adult stem cells. They possess highly similar histological characteristics to human tissues and can reproduce some physiological functions of organs. Organoid culture provides a new platform for drug screening and complements existing 2D culture methods and animal model systems with high information content. Currently, organoids can be used to screen drugs including chemotherapy drugs, small molecule targeted drugs, and antibody drugs. The core indicators for drug screening are typically IC50 and cell inhibition rate. Based on these indicators, drugs with the best tumor-suppressing effects are selected from the screened drugs.

[0005] After successfully culturing organoids, their evaluation and validation are crucial. This is typically achieved through methods such as gene sequencing, immunofluorescence, and hematoxylin and eosin (HE) staining, identifying organoids from multiple dimensions including morphology, histopathology, and molecular genetics. The main purpose of the evaluation is to determine the consistency between the organoids and the prototum, facilitating subsequent drug screening.

[0006] Existing technologies disclose a culture medium specifically for small cell lung cancer organoids. However, experimental results show that organoid growth is poor under these conditions, with small organoid volume, slow growth, and unclear cell passageability. Furthermore, the growth and passage activity of the cultured organoids was not tested, making the maintenance of tumor characteristics uncertain. The cultured small cell lung cancer organoids are derived from small cell lung cancer patients, and based on experience with existing technologies, tumor gene characteristics of patient-derived lung cancer organoids are unstable and easily lost during long-term in vitro culture and passage. Another existing technology discloses a culture medium system and method for lung cancer organoids targeting lung adenocarcinoma, but this medium is not specifically designed for small cell lung cancer. Therefore, existing small cell organoid culture methods still have many problems, such as poor organoid growth, small size, slow growth, and a lack of organoid sources and culture conditions suitable for long-term maintenance of tumor characteristics. These problems limit the application of organoid culture technology in studying tumor heterogeneity and radiotherapy / chemotherapy resistance mechanisms in small cell lung cancer.

[0007] Therefore, there is an urgent need in this field to develop novel culture media and methods specifically for small cell lung cancer tumor organoids, so as to achieve large and rapid growth of organoid cells, continuous in vitro culture of small cell lung cancer organoids, and better maintenance of SCLC tumor invasive characteristics. Summary of the Invention

[0008] To address one of the aforementioned technical problems in the prior art, this invention provides a culture medium and culture method suitable for the culture of small cell lung cancer organoids with a specific gene background (Trp53 / Rb1 / Pten gene mutation), which enables continuous passage culture of small cell lung cancer organoids and maintains the tumor characteristics of small cell lung cancer.

[0009] In a first aspect, the present invention provides a culture medium for small cell lung cancer organoids, comprising a basal culture medium, an antimicrobial agent, a culture medium supplement, cytokines, and optionally a buffer.

[0010] In some implementations, small cell lung cancer is Trp53 / Rb1 / Pten gene-mutant small cell lung cancer.

[0011] In some preferred embodiments, the small cell lung cancer is Trp53 / Rb1 / Pten gene deletion type small cell lung cancer.

[0012] In some embodiments, the basal culture medium is selected from MEM, BME, RPMI-1640, DMEM, DMEM / F12 or Advanced DMEM / F12 medium, preferably DMEM / F12 or Advanced DMEM / F12 medium, more preferably Advanced DMEM / F12 medium.

[0013] In some embodiments, the antimicrobial agent is selected from penicillin, streptomycin, aminoglycoside antibiotics, amphotericin B, ornidazole, primocin, or any combination thereof.

[0014] In some preferred embodiments, the antimicrobial agent includes penicillin, streptomycin, and primocin.

[0015] In some embodiments, the culture medium supplement comprises one or more of the following: fetal bovine serum, newborn calf serum, human serum, B27 supplement, N2 supplement, amino acids selected from L-glutamine, N-acetylcysteine, nicotinamide, GlutaMAX, or any combination thereof, and ascorbic acid; preferably, the culture medium supplement comprises B27 supplement, N-acetylcysteine, and GlutaMAX.

[0016] In some embodiments, the cytokines are selected from WNT signaling pathway activators, bone morphogenetic protein inhibitors, epidermal growth factor, fibroblast growth factor, transforming growth factor, vascular endothelial growth factor, hepatocyte growth factor, bone morphogenetic protein, neuromodulatory proteins, ROCK inhibitors, transforming growth factor β inhibitors, p38 MAPK inhibitors, MDM2 inhibitors, or any combination thereof. In some preferred embodiments, the cytokines comprise WNT signaling pathway activators, bone morphogenetic protein inhibitors, fibroblast growth factor, ROCK inhibitors, transforming growth factor β inhibitors, and p38 MAPK inhibitors.

[0017] In some embodiments, the WNT signaling pathway activator comprises a GSK3β inhibitor, R-spondin protein, Wnt protein, or any combination thereof. In some preferred embodiments, the WNT signaling pathway activator is selected from a combination of a GSK3β inhibitor and R-spondin protein.

[0018] In some embodiments, the bone morphogenetic protein inhibitor is selected from Noggin, ML347, DMH1, LDN-212854, LDN-214117, LDN-193189, Dorsomorphin, and osteosclerosing protein or any combination thereof, with Noggin being preferred.

[0019] In some embodiments, the fibroblast growth factor is selected from fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 7 (FGF7), fibroblast growth factor 9 (FGF9), fibroblast growth factor 10 (FGF10), or any combination thereof, preferably FGF2.

[0020] In some embodiments, the vascular endothelial growth factor is selected from vascular endothelial growth factor A, vascular endothelial growth factor B, vascular endothelial growth factor C, or any combination thereof.

[0021] In some embodiments, the ROCK inhibitor is selected from Y-27632, Fasudi HCl, Thiazovin, GSK42928A, ZINC00881524, RKI-1447, AT13148, Belumosudil, GSK269962A HCl, Chroman 1, or any combination thereof, with Y-27632 being preferred.

[0022] In some embodiments, the transforming growth factor β inhibitor is selected from RepSox, E-616452, A83-01, A77-01, SB431542, GW788388, TGFβRI-IN-2, BIBF0775, ITD1, SJN2511, D4476, LY364947, SB505124, SB525334, SD208, Activin A, or any combination thereof, preferably A83-01.

[0023] In some embodiments, the p38 MAPK inhibitor is selected from SB203580, Doramimod (BIRB796), SB202190, LY2228820, VX-702, PH-797804, VX-745, TAK-71599, BMS-582949 or any combination thereof, with SB202190 being preferred.

[0024] In some embodiments, the MDM2 inhibitor is selected from Rebemadlin, Idasanutlin, Nutlin-3, Nutlin-3a, Nutlin-3b, MX69, NVP-CGM097, MI-773 (SAR405838), RG-7112, HDM201 (Siremadlin), YH239-EE, NSC207895, Serdemetan (JNJ-26854165), or any combination thereof.

[0025] In some embodiments, the GSK3β inhibitor is selected from CHIR-99021, GSK3β inhibitor IX, GSK3β inhibitor VII, Indirubin, L803-mts, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-Azakenpaullone, SB216763, LY2090314, CHIR-98014, BIO-acetoxime, SAR502250, trihydrochloride, or any combination thereof, with CHIR-99021 being preferred.

[0026] In some embodiments, R-spondin is selected from R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4 or any combination thereof, with R-spondin 1 being preferred.

[0027] In some embodiments, the Wnt protein is selected from Wnt1, Wnt2, Wnt2b (Wnt13), Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a (Wnt14), Wnt9b (Wnt14b), Wnt10a, Wnt10b, Wnt11, and Wnt16, or any combination thereof.

[0028] In some embodiments, the culture medium further comprises a buffer selected from bicarbonate, HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), HEPPSO (3-(hydroxyethylpiperazine)-2-hydroxypropanesulfonic acid), MOPS (3-morpholinopropanesulfonic acid), POPSO (piperazine-N,N'-bis(2-hydroxypropanesulfonic acid)), PIPES (piperazine-1,4-diethanesulfonic acid), DIPSO (3-[NN-bis(2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid), MES (2-morpholinoethanesulfonic acid), TES (Tris ethanesulfonic acid), BES (N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid), or any combination thereof, preferably HEPES.

[0029] In some preferred embodiments, the small cell lung cancer organoid culture medium comprises:

[0030] The basal culture medium, wherein the basal culture medium is selected from Advanced DMEM / F12 medium;

[0031] An antimicrobial agent, wherein the antimicrobial agent is selected from penicillin / streptomycin bispecific antibiotics, primocin, or a combination thereof;

[0032] Culture medium supplement, wherein the culture medium supplement is selected from B27 supplement, N-acetylcysteine, GlutaMAX, or any combination thereof;

[0033] Cytokines, wherein the cytokines are selected from Noggin, R-spondin 1, CHIR-99021, FGF2, Y-27632, A83-01, SB202190 or combinations thereof, preferably, the cytokines are composed of Noggin, R-spondin 1, CHIR-99021, FGF2, Y-27632, A83-01 and SB202190;

[0034] And a buffer, wherein the buffer is selected from HEPES.

[0035] In some embodiments, the basal culture medium may be 0.7× to 2× (e.g., 0.7×, 1×, 1.5×, 2× or any value between them) as needed, preferably 1×.

[0036] In some embodiments, the concentration range of the antimicrobial agent, such as penicillin / streptomycin bispecific antibody, is 0.5% to 3% (e.g., 0.5%, 1%, 2%, 3% or any value between these).

[0037] In some embodiments, the concentration range of the antimicrobial agent, such as Primocin, is 10 to 80 ng / mL (e.g., 10 ng / mL, 20 ng / mL, 30 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL or any value between these ranges), preferably 30 to 60 ng / mL.

[0038] In some embodiments, the concentration range of the culture medium supplement, such as B27 supplement, is 0.5 to 10 mM (e.g., 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM or any value in between), preferably 1 to 3 mM.

[0039] In some embodiments, the concentration of the culture medium supplement, such as N-acetylcysteine, is in the range of 0.1 to 5 mM (e.g., 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM or any value between these ranges), preferably 0.5 to 2 mM.

[0040] In some embodiments, the concentration range of the bone morphogenetic protein inhibitor, such as Noggin, is 50 to 200 ng / mL (e.g., 50 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 110 ng / mL, 120 ng / mL, 150 ng / mL, 200 ng / mL or any value between these ranges), preferably 80 to 120 ng / mL.

[0041] In some embodiments, the concentration range of the WNT signaling pathway activator is 100 to 1000 ng / mL (e.g., 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, 1000 ng / mL or any value between these ranges), preferably 300 to 600 ng / mL.

[0042] In some embodiments, the concentration range of the WNT signaling pathway activator is 1 to 10 μM (e.g., 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM or any value between these ranges), preferably 2 to 6 μM.

[0043] In some embodiments, the small cell lung cancer organoid culture medium comprises a combination of R-Spondin protein and a GSK3β inhibitor. In some embodiments, the concentration of R-Spondin protein, such as R-spondin 1, ranges from 100 to 1000 ng / mL (e.g., 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, 500 ng / mL, 600 ng / mL, 700 ng / mL, 800 ng / mL, 900 ng / mL, 1000 ng / mL or any value between these values), preferably 300 to 600 ng / mL. In some embodiments, the concentration of GSK3β inhibitor, such as CHIR-99021, ranges from 1 to 10 μM (e.g., 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM or any value between these values), preferably 2 to 6 μM.

[0044] In some embodiments, the concentration of fibroblast growth factor is in the range of 2 to 10 ng / mL (e.g., 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL or any value between these), preferably 3 to 8 ng / mL.

[0045] In some embodiments, the concentration range of the ROCK inhibitor is 1 to 10 μM (e.g., 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM or any value between these ranges), preferably 5 to 10 μM.

[0046] In some embodiments, the concentration range of the transforming growth factor β inhibitor is 500 to 2000 nM (e.g., 500 nM, 750 nM, 1000 nM, 1250 nM, 1500 nM, 1750 nM, 2000 nM or any value between these ranges), preferably 300 to 800 nM.

[0047] In some embodiments, the concentration range of the p38 MAPK inhibitor is 1–10 μM (e.g., 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, etc.), preferably 3–10 μM.

[0048] In some embodiments, the small cell lung cancer organoid culture medium contains a buffer such as HEPES, the concentration of which is in the range of 5 to 20 mM (e.g., 5 mM, 8 mM, 11 mM, 14 mM, 16 mM, 18 mM, 20 mM or any value in between), preferably 8 to 15 mM.

[0049] In some preferred embodiments, the small cell lung cancer organoid culture medium comprises the following components at concentrations: GlutaMAX, 0.5–10 mM; HEPES, 5–20 mM; penicillin / streptomycin (P / S antibiotic), 0.5–3%; Primocin, 10–80 ng / mL; Noggin, 50–200 ng / mL; B27 supplement, 0.5–10 mM; N-acetylcysteine, 0.1–5 mM; R-Spondin1, 100–1000 ng / mL; CHIR-99021, 1–10 μM; FGF2, 2–10 ng / mL; Y-27632, 1–10 μM; A83-01, 300–2000 nM; SB202190, 1–10 μM.

[0050] In some preferred embodiments, the small cell lung cancer organoid culture medium comprises the following components at concentrations: GlutaMAX, 1–3 mM; HEPES, 8–15 mM; P / S antibiotics, 0.5–3%; Primocin, 30–60 ng / mL; Noggin, 80–120 ng / mL; B27 supplement, 1–3 mM; N-acetylcysteine, 0.5–2 mM; R-Spondin 1, 300–600 ng / mL; CHIR-99021, 2–6 μM; FGF2, 3–8 ng / mL; Y-27632, 5–10 μM; A83-01, 500–800 nM; SB202190, 3–10 μM.

[0051] Secondly, the present invention provides a method for culturing small cell lung cancer organoids, comprising the following steps: adding the small cell lung cancer organoid culture medium of the first aspect to a three-dimensional scaffold containing tumor cells and / or tumor cell clusters of small cell lung cancer. In some embodiments, the three-dimensional scaffold is selected from synthetic biological scaffolds, natural scaffolds, or any combination thereof.

[0052] In some embodiments, the synthetic biological scaffold is selected from porous solids, nanofibers, or hydrogels.

[0053] In some implementations, small cell lung cancer refers to small cell lung cancer with Trp53 / Rb1 / Pten gene mutations.

[0054] In some preferred embodiments, the small cell lung cancer is Trp53 / Rb1 / Pten gene deletion type small cell lung cancer.

[0055] In some embodiments, tumor cells and / or clusters of tumor cells containing small cell lung cancer are obtained by contacting digestive fluid with small cell lung cancer tumor tissue.

[0056] In some embodiments, the digestive fluid is lung tissue digestive fluid, wherein the lung tissue digestive fluid may contain one or more of the following: collagenase, calcium chloride, zinc chloride, DNase, neutral protease, fetal bovine serum, Liberase enzyme, antibiotics, preferably DNase I.

[0057] In some embodiments, the collagenase is selected from collagenase I, collagenase II, collagenase III, collagenase IV, collagenase V or any combination thereof, with collagenase I being preferred.

[0058] In some implementations, the antibiotic is a penicillin / streptomycin bispecific antibody.

[0059] In some preferred embodiments, the lung tissue digestion fluid contains collagenase I, DNAase I, and penicillin / streptomycin bispecific antibodies.

[0060] In some embodiments, the lung tissue digestion fluid contains collagenase, such as collagenase I, in a concentration range of 0.1 mg / mL to 5 mg / mL (e.g., 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL or any value between these ranges), preferably 1 mg / mL.

[0061] In some embodiments, the lung tissue digestion fluid contains a DNase, such as DNase I, at a concentration ranging from 0.1 to 0.5 mg / mL (e.g., 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, or any value between these values).

[0062] In some embodiments, the lung tissue digestive fluid contains an antibiotic, such as penicillin / streptomycin, at a concentration ranging from 0.5% to 3% (e.g., 0.5%, 1%, 2%, 3% or any value between these), preferably 1%.

[0063] In some preferred embodiments, the lung tissue digestion solution contains 1 mg / mL collagenase I, 0.1–0.5 mg / mL DNase I, and 1% penicillin / streptomycin antibiotics. This lung tissue digestion solution is more suitable for digesting and isolating small cell lung cancer cells with Trp53 / Rb1 / Pten gene mutations, exhibiting high cell viability and a high success rate in organoid culture.

[0064] In some embodiments, the digestion temperature is 37°C, and the digestion time is 30 minutes to 3 hours (e.g., 30 minutes, 1 hour, 2 hours, 3 hours, etc.), preferably 1 hour. These digestion conditions are suitable for the isolation of small cell lung cancer cells and can better maintain tumor cell viability, which is beneficial for the survival of subsequent organoids.

[0065] In some implementations, the method for culturing small cell lung cancer organoids also includes changing the medium every 3 to 4 days during the culture process, and passage of the organoids when the density of small cell lung cancer organoids reaches 80%.

[0066] Thirdly, the present invention provides a small cell lung cancer organoid obtained by the culture method of the second aspect.

[0067] Fourthly, the present invention provides an organoid for small cell lung cancer with a Trp53 / Rb1 / Pten gene mutation.

[0068] In some embodiments, small cell lung cancer organoids with Trp53 / Rb1 / Pten gene mutations exhibit multiple morphologies after being passaged to the first generation and beyond, wherein the multiple morphologies are selected from two or more of the following: solid, budding, vesicular, and mixed forms.

[0069] In some implementations, organoids of small cell lung cancer with Trp53 / Rb1 / Pten gene mutations are obtained through the culture method of the second aspect.

[0070] In some preferred embodiments, the small cell lung cancer is Trp53 / Rb1 / Pten gene deletion type small cell lung cancer.

[0071] Fifthly, the present invention provides a use of small cell lung cancer organoids in the third or fourth aspect, including drug toxicity testing, discovery, screening and validation of new drugs.

[0072] In a sixth aspect, the present invention provides a small cell lung cancer organoid chip, which includes the small cell lung cancer organoids of the third or fourth aspect.

[0073] In some embodiments, the small cell lung cancer organoid chip is a three-dimensional respiratory chip that can simulate the in vivo lung respiratory physiological microenvironment.

[0074] In some embodiments, the three-dimensional respiratory chip is homemade; in others, it is commercially available.

[0075] In some embodiments, the material of the three-dimensional breathing chip is selected from polydimethylsiloxane (PDMS), glass, silicon, plexiglass, paper, polycaprolactone (PCL), polyurethane (TPU), or any combination thereof.

[0076] In some implementations, the chip body is fabricated using methods such as photolithography, molding, micro-contact embossing, laser etching, machining, and 3D printing.

[0077] In a seventh aspect, the present invention provides a method for screening targeted drugs for small cell lung cancer, comprising: contacting a small cell lung cancer organoid chip (as described in the third or fourth aspect or the fifth aspect) with a candidate drug.

[0078] Beneficial effects of the present invention

[0079] 1. The organoid culture medium for small cell lung cancer of this invention enables the continuous and stable culture and passage of organoids from small cell lung cancer with a specific gene background (Trp53 / Rb1 / Pten gene mutants). Through optimization of the culture medium and method, the combined use of Noggin and CHIR-99021 achieves better culture results, resulting in large-volume, well-grown, and fast-growing small cell lung cancer organoids, reducing the difficulty of organoid culture. The specific and targeted composition of the culture medium leads to more diverse organoid morphologies, stable passage while maintaining stable tumor gene characteristics, and effectively preserving the invasiveness and heterogeneity of small cell lung cancer tumors under the specific gene background, thus possessing greater clinical application value. This solves the problems of poor organoid growth and slow growth rate in existing technologies, and has excellent application prospects.

[0080] 2. In addition, this invention also obtained organoid models or organoid microarrays of small cell lung cancer with specific gene backgrounds (Trp53 / Rb1 / Pten gene mutations), which can reproduce the occurrence and development of small cell lung cancer in vitro, provide a tumor microenvironment that is closer to physiology, simulate the characteristics of real SCLC, and can be used for the detection of drug sensitivity in clinical oncology patients and the testing and application of potential effective target drugs, providing a powerful tool for convenient and rapid targeted drug screening and even personalized precision treatment of oncology patients. Attached Figure Description

[0081] Figure 1 The process of constructing organoids from Trp53 / Rb1 / Pten-deficient small cell lung cancer is shown.

[0082] Figure 2 The images show Trp53 / Rb1 / Pten-deficient mouse small cells stained with hematoxylin / eosin to determine lung cancer tumorigenesis.

[0083] Figure 3 The in situ distribution of Td-tomato-positive SCLC cells in Trp53 / Rb1 / Pten-deficient mice and the digestion and isolation of lung tumor cells are shown.

[0084] Figure 4 The growth of Td-tomato-positive SCLC organoids from Trp53 / Rb1 / Pten deletion mice is shown from passage 0 to day 3.

[0085] Figure 5 The growth morphological changes of Td-tomato-positive SCLC organoids from Trp53 / Rb1 / Pten-deficient mice are shown from the first generation culture to day 4.

[0086] Figure 6The study showed that on day 5 of second-generation culture of Td-tomato-positive SCLC organoids from Trp53 / Rb1 / Pten-deficient mice, cells exhibited a highly invasive and protruding morphology.

[0087] Figure 7 The study showed that Td-tomato-positive SCLC organoids from Trp53 / Rb1 / Pten-deficient mice exhibited polymorphic phenotypes on day 15 of second-generation culture.

[0088] Figure 8 The study showed that Td-tomato-positive SCLC organoids from Trp53 / Rb1 / Pten-deficient mice exhibited polymorphic phenotypes on day 12 of the fourth generation culture.

[0089] Figure 9 The study showed that Td-tomato-positive SCLC organoids from Trp53 / Rb1 / Pten-deficient mice exhibited cell migration and highly invasive morphology on day 10 after 5th generation culture.

[0090] Figure 10 The study showed neuroendocrine cell markers and Trp53 / Rb1 / Pten gene mRNA levels in Td-tomato-positive SCLC organoids.

[0091] Figure 11 The growth of Td-tomato-positive SCLC organoids from Trp53 / Rb1 / Pten-deficient mice is shown in media without Noggin or CHIR-99021. Detailed Implementation

[0092] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. Such structures and techniques have also been described in many publications.

[0093] definition

[0094] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0095] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0096] The term "mammal" as used herein refers to humans and other mammals. Mammals other than humans may include: rodents such as mice, rats, guinea pigs, and hamsters; non-human primates such as chimpanzees; even-toed ungulates such as cattle, goats, and sheep; perissodactyls such as horses; and pets such as rabbits, dogs, and cats. In some embodiments, the mammal is a rodent or a non-human primate. In one embodiment, the mammal is a human.

[0097] The term "organoid" as used in this article refers to cell clusters derived from stem cells or organ tissues that, after being cultured in vitro in three dimensions, can differentiate and self-organize to form some specific functions and structures of corresponding human organs.

[0098] The term "small cell lung cancer" as used in this article refers to a malignant epithelial tumor composed of small cells. Small cell lung cancer (SCLC) accounts for 15%–20% of all lung cancers and is divided into four molecular subtypes: SCLC-A, SCLC-N, SCLC-P, and SCLC-Y, characterized by the expression of transcription factors ASCL1, NEUROD1, POU2F3, and YAP1, respectively. Somatic mutations and copy number deletions of Trp53 and RB1 are the main gene variations in small cell lung cancer.

[0099] The terms "small cell lung cancer organoids" or "small cell lung cancer tumor organoids" used in this article have the same meaning. They refer to a three-dimensional model that is highly similar to the tumor characteristics of the patient, which is established by using an in vitro three-dimensional (3D) cell culture system from fresh tumor tissue from small cell lung cancer patients or tumor tissue from small cell lung cancer animal models induced in vivo.

[0100] The term "Trp53" (Transformation-related protein 53) used in this article refers to a tumor suppressor gene encoding the p53 protein. Trp53 is a tumor suppressor protein and transcription factor that regulates cell division, prevents cells with mutated or damaged DNA from dividing, and transmits apoptosis signals to these cells through transcriptional regulation, thereby preventing tumor formation. The Trp53 (mouse) / TP53 (human) gene is altered in approximately 90% of small cell lung cancer (SCLC) and approximately 50% of non-small cell lung cancer (NSCLC).

[0101] In this article, "RB1" (RetinoBlastoma 1, RB1) refers to a tumor suppressor gene encoding a 150kD nuclear phosphorylated protein. This protein inhibits cell proliferation and transformation, and its phosphorylation state is the main form by which the RB gene regulates cell growth and differentiation. Abnormalities in the RB1 gene are mainly manifested as allele deletions and gene mutations, leading to abnormalities in both mRNA and protein.

[0102] The term "PTEN" (Phosphatase and Tensin Homolog) used in this article refers to the tumor suppressor gene encoding the classic PTEN protein, composed of 403 amino acids. The PTEN protein possesses phosphatase activity and can inhibit tumor development and progression by antagonizing the activity of phosphorylases such as tyrosine kinases. The PTEN gene is primarily inactivated through allele deletion, gene mutation, and methylation.

[0103] The term "culture medium" as used in this article refers to a medium containing the components required for cell culture. Culture media can contain basal medium and various additives. Culture media can be prepared by combining basal medium with additives. Culture media can further contain any one or both of extracellular matrix and substances that aid cell proliferation.

[0104] The term "basal medium" as used herein can refer to any known culture medium used for cell culture, such as Durbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Eagle Basal Medium (BME), RPMI-1640, KnockOut DMEM, KnockOut DMEM / F12, neurobasal, or DMEM / F12. In one embodiment, the basal medium is DMEM / F12. In one embodiment, the basal medium is Advanced DMEM / F12. In one embodiment, the basal medium comprises combinations of the above-described exemplary basal media in various proportions.

[0105] The term "Primocin" as used in this article refers to: primary cell antibiotics (such as Primocin). TMInvivoGen's #Ant-pm-1 (or other products with the same composition) is an antibiotic that kills Gram-positive bacteria, Gram-negative bacteria, mycoplasma, and fungi. It effectively prevents bacterial, mycoplasma, and fungal contamination while minimizing cytotoxicity because its targets are only found on microorganisms. Specifically, its targets for bacteria are DNA helicase and the 30S and 50S ribosome subunits of prokaryotic cells; and for fungi, it is ergosterol (a molecule found only in the cell membranes of fungi and yeast).

[0106] The "GlutaMAX" used in this article refers to: GlutaMAX TM Supplement, composed of L-alanyl-L-glutamine dipeptide, is a substitute for L-glutamine. It has a concentration of 200 mM and is produced in 0.85% NaCl solution.

[0107] The organoid culture medium of the present invention further comprises at least one amino acid and its prodrug. In some embodiments, the amino acid is selected from L-glutamine, N-acetylcysteine, nicotinamide, GlutaMAX, or combinations thereof. In organoid culture, it exhibits antioxidant activity and inhibits neuronal apoptosis.

[0108] As used in this article, "cytokines" refers to a combination of two or more substances that affect the efficiency of organoid formation from lung epithelial cells or lung cancer cells. For example, cytokines can be a combination of two or more substances that improve the efficiency of organoid formation from lung epithelial cells or lung cancer cells and / or cytokines can contain substances that maintain the differentiation capacity of the constituent cells of the formed organoids or induce differentiation into specific cell species.

[0109] In some embodiments, the cytokines comprise at least one selected from keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), fibroblast growth factor 10 (FGF10), and fibroblast growth factor 2 (FGF2), a combination of a bone morphogenetic protein (BMP) inhibitor (e.g., Noggin) and a TGFβ inhibitor (e.g., A83-01); or, preferably, also contain a WNT activator (e.g., CHIR99021 or R-Spondin 1). In some embodiments, a ROCK inhibitor (e.g., Y-27632) is also included. In some embodiments, a p38 MAPK inhibitor (e.g., SB202190) is also included. In some embodiments, an MDM2 inhibitor is also included. In some embodiments, a natural or synthetic substance with similar properties known in the art, such as dexamethasone, is also included.

[0110] The term "BMP (bone morphogenetic protein) inhibitor" as used in this article refers to substances that bind to BMP molecules to form a complex. BMP inhibitors can be low-molecular-weight compounds, proteins (e.g., antibodies), DNA, RNA, low-molecular-weight interfering RNA, or antisense oligonucleotides. BMP inhibitors are commercially available. BMP inhibitors can be, but are not limited to, Noggin (a BMP inhibitor), ML347, DMH1, LDN-212854, LDN-214117 (a BMP receptor-selective inhibitor), LDN-193189, Dorsomorphin (a small molecule BMP inhibitor), and sclerostin, or any combination thereof.

[0111] As used herein, "WNT activator" refers to a molecule that partially or completely enhances, increases, or stimulates the biological activity of WNT proteins. Suitable WNT activators include, but are not limited to, proteins, nucleic acids, small molecules, or combinations thereof. In some embodiments, the WNT activator is selected from GSK3β inhibitors, R-spondin, and Wnt proteins, or any combination thereof; in some embodiments, the GSK3β inhibitor is selected from CHIR-99021, GSK3β inhibitor IX, GSK3β inhibitor VII, Indirubin, L803-mts, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-Azakenpaullone, SB216763, or any combination thereof; and the R-spondin is selected from R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4, or any combination thereof. In organoid culture, cell differentiation can be induced.

[0112] The term "ROCK inhibitor" as used in this article refers to an inhibitor of Rho-associated coil-containing protein kinase (ROCK), which inhibits ROCK1 and ROCK2 by competing with ATP for binding to catalytic sites. ROCK inhibitors include, but are not limited to, proteins, nucleic acids, small molecules, or combinations thereof. In some embodiments, the ROCK inhibitor is selected from Y-27632, Fasudi HCl, Thiazovivin, GSK42928A, ZINC00881524, RKI-1447, AT13148, Belumosudil, GSK269962A HCl, Chroman1, or combinations thereof. In organoid culture, it can inhibit stem cell apoptosis, improve cloning efficiency, and prolong cell passage.

[0113] The term "p38 MAPK inhibitor" as used herein refers to a p38 MAP kinase (MAPK) inhibitor. p38 MAPK is the mammalian homologue of the yeast HOG kinase and plays an important role in cytokine and stress-induced signal transduction. p38 MAPK inhibitors include, but are not limited to, chemical inhibitors, neutralizing antibodies, aptamers, ligand trapping agents, antisense nucleotides, protein inhibitors, and engineered peptides. In some embodiments, the p38 MAPK inhibitor is selected from SB203580, Doramimod (BIRB796), SB202190, LY2228820, VX-702 (inhibiting p38a MAPK), PH-797804, VX-745 (acting on p38a), TAK-71599 (acting on p38a), BMS-582949 (inhibiting p38a MAPK), or any combination thereof.

[0114] The term "transforming growth factor β inhibitor" as used in this article refers to inhibitors of the TGF-β superfamily that regulate cell growth and differentiation. In some embodiments, the TGF-β inhibitor is selected from RepSox, E-616452, A83-01, A77-01, SB431542, GW788388, TGFβRI-IN-2, BIBF0775, ITD1, SJN2511, D4476, LY364947, SB505124, SB525334, SD208, or any combination thereof.

[0115] The combined use of Y-27632, A8301 and CHIR 99021 can improve cell survival rate and cell proliferation capacity.

[0116] As used in this article, "MDM2 inhibitor" refers to an inhibitor of murine double minute 2 (MDM2). The MDM2 protein was initially identified as an oncogene in mouse transformation systems. MDM2 functions by binding to p53 and blocking p53-mediated reverse transcription activation of co-transfected reporter gene structures. In some embodiments, the MDM2 inhibitor is selected from Rebemadlin, Idasanutlin, Nutlin-3, Nutlin-3a, Nutlin-3b, MX69, NVP-CGM097, MI-773 (SAR405838), RG-7112, HDM201 (Siremadlin), YH239-EE, NSC207895, Serdemetan (JNJ-26854165), or any combination thereof.

[0117] The “HEPES” used in this article refers to (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), an amphoteric organic chemical buffer, which is a type of “Good” buffer and is used as a buffering reagent in cell culture media.

[0118] As used herein, "Matrigel or extracellular matrix" refers to a three-dimensional matrix that can polymerize at room temperature to form a biologically active matrix that mimics the structure, composition, physical properties, and function of the in vivo cell basement membrane, thus facilitating cell culture and differentiation in vitro. The extracellular matrix is ​​commercially available, and commercially available extracellular matrices may include, for example, extracellular matrix proteins (Invitrogen), basement membrane preparations derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells (e.g., Cultrex Basement Membrane Extract (Trevigen), or Matrigel (Corning)). The extracellular matrix (ECM) may be a synthetic extracellular matrix (e.g., ProNectin (Sigma Z378666)). The extracellular matrix may be one or a mixture of two or more. In one embodiment, the extracellular matrix is ​​Matrigel. In one embodiment, the culture medium does not contain an extracellular matrix. In some embodiments, the matrix is ​​selected from one or any combination of standard matrix, low-growth-factor matrix, phenol red-free matrix, and high-concentration matrix.

[0119] The term "collagenase" as used herein refers to collagenase, an enzyme that hydrolyzes proline in the intercellular matrix, thereby dissociating cells. Collagenase has a strong digestive effect on collagen, acting primarily on the intercellular matrix with minimal damage to cells, making it suitable for digesting and separating fibrous tissue and harder cancerous tissue. Calcium and magnesium ions and serum do not affect the activity or digestive effect of collagenase. In some embodiments, the collagenase is selected from collagenase I, collagenase II, collagenase III, collagenase IV, collagenase V, or any combination thereof.

[0120] As used herein, the term "organ-on-a-chip" refers to devices fabricated using microscale engineering techniques, such as microfluidic chips, to cultivate organoids, thereby controlling and regulating organoid development. Microfluidic technology is used to control liquid flow, combined with cell-cell interactions, matrix properties, and biochemical and biomechanical characteristics, to construct three-dimensional organ physiological microsystems on a chip. In some embodiments, the organ-on-a-chip includes a chip body and cells (or micro-tissues); in some embodiments, the chip body is commercially available from companies such as Emulate; in other embodiments, the chip body is self-made using manufacturing methods known in the art. For example, it is fabricated using conventional methods of microfluidic chip technology based on MEMS (Micro-Electro-Mechanical System) fabrication processes. Materials used in self-made chips include, but are not limited to, polydimethylsiloxane (PDMS), glass, silicon, plexiglass, paper, polycaprolactone (PCL), and polyurethane (TPU). In some embodiments, the chip body fabrication methods include, but are not limited to, photolithography, molding, micro-contact imprinting, laser etching, machining, and 3D printing.

[0121] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0122] Example

[0123] Unless otherwise specified, the experimental methods in the embodiments of this invention are all conventional methods.

[0124] Unless otherwise specified, the experimental materials used in the embodiments of this invention are all commercially available conventional biochemical reagents.

[0125] The main experimental reagents involved in the following examples are as follows:

[0126] Advanced DMEM / F12 medium, Invitrogen, catalog number: 12634010;

[0127] Penicillin / streptomycin (P / S) bispecific antibody, Invitrogen, catalog number: 15140-148;

[0128] Glutamine (GlutaMAX), Invitrogen, product number: 35050061;

[0129] Primary cell antibiotic (Primocin), InvivoGen, catalog number: Ant-pm-1;

[0130] HEPES buffer, Invitrogen, catalog number: H1095;

[0131] N-acetylcysteine, Sigma, product number: A9165;

[0132] B27 supplement, Invitrogen, product number: 17504044;

[0133] Recombinant human Noggin protein, Novoprotein, catalog number: C028;

[0134] Recombinant human R-spondin 1 protein, Novoprotein, catalog number: CX83;

[0135] GSK-3α / β inhibitor (CHIR-99021), Selleck, catalog number: S2924;

[0136] Fibroblast growth factor 2 (FGF2), Peprotech, catalog number: 100-18B-50;

[0137] ROCK1 / 2 inhibitor (Y-27632), MCE Corporation, catalog number: HY-10071;

[0138] TGF-β receptor inhibitor (A83-01), MCE Corporation, catalog number: HY-10432;

[0139] p38 MAPK inhibitor (SB202190), MCE Corporation, catalog number: HY-10295;

[0140] Matrigel, Corning, part number: 356231;

[0141] Type I collagenase, Invitrogen, catalog number: 17100017.

[0142] Example 1. Construction of Cgrp-CreERT2; Trp53f / f; Rb1f / f; Ptenf / f; Rosa26-Td-tomato mice

[0143] Cgrp-CreERT2; Trp53 f / f； Rb1 f / f Pten f / f The Rosa26-Td-tomato mouse was constructed as follows: Trp53 was constructed. f / f； Rb1 f / f Pten f / fRosa26-Td-tomato mice, when bred with Cgrp-CreERT2 mice, produce Cgrp-CreERT2; Trp53 f / f； Rb1 f / f Pten f / f Rosa26-Td-tomato conditional knockout mice were induced. Two-month-old mice were intraperitoneally injected with Tamoxifen for three consecutive days to induce specific knockout of the Trp53 / Rb1 / Pten gene in pulmonary neuroendocrine cells. Two months later, small cell lung cancer (SCLC) spontaneously developed in the mice. Lung tissue was isolated from the mice, and pathological analysis was performed to determine the formation of SCLC tumors. Western blotting was used to verify the knockout of the Trp53 / Rb1 / Pten gene in tumor cells.

[0144] Example 2. Preparation of organoid culture medium for Trp53 / Rb1 / Pten-deficient small cell lung cancer

[0145] The culture medium consisted of Advanced DMEM / F12 medium, GlutaMAX, HEPES buffer, penicillin / streptomycin antibiotics, primary cell antibiotic (Primocin), recombinant human Noggin protein, recombinant human R-spondin 1 protein, B27 supplement, N-acetylcysteine, GSK-3α / β inhibitor (CHIR-99021), fibroblast growth factor 2 (FGF2), ROCK1 / 2 inhibitor (Y-27632), TGF-β receptor inhibitor (A83-01), and p38 MAPK inhibitor (SB202190). The concentrations used are shown in Table 1.

[0146] Table 1. Culture medium components

[0147]

[0148] Example 3. Culture method for organoids from Trp53 / Rb1 / Pten-deficient small cell lung cancer

[0149] The flowchart of organoid culture method for mouse Trp53 / Rb1 / Pten deletion small cell lung cancer is as follows: Figure 1 As shown, the specific steps are as follows:

[0150] (1) Obtain Trp53 / Rb1 / Pten-deficient small cell lung cancer tissue. Cgrp-CreERT2; Trp53 f / f； Rb1 f / f Pten f / fTwo months after Rosa26-Td-tomato mice were intraperitoneally injected with tamoxifen, their respiratory distress was monitored in real time. Further CT scans confirmed tumor formation. The mice were then euthanized by cervical dislocation, placed in 10cm cell culture dishes, and sprayed with 75% alcohol. Lung tissue was then separated in a clean bench, washed 2-3 times in pre-cooled DPBS buffer, and the distribution of red (Td-tomato positive) SCLC tumor cells was observed under a microscope. Figure 3 Subsequently, a suitable amount of lung tissue was collected and frozen at -80°C for subsequent pathological examination. Figure 2 DNA, RNA and protein analysis.

[0151] (2) Enzymatic digestion of small cell lung cancer tissue. Prepare 10 mL of lung tissue digestion solution (1 mg / mL type I collagenase, 0.1–0.5 mg / mL DNase I, 1% penicillin / streptomycin) in a 15 mL centrifuge tube. Transfer 2 mL of the digestion solution to a 6 cm cell culture dish. Immerse the remaining lung tumor tissue in the digestion solution and use a sterilized scalpel to cut it to a depth of 1–2 mm. 3 Small tissue fragments were collected from the lung tissue. The suspension was added back to the original digestion solution and digested at low speed in a 37°C oven for 1 hour. After digestion, the centrifuge tube was removed and manually shaken vigorously for 15 seconds. The digested cell suspension was then filtered through a 70μm filter, and the filtrate containing SCLC tumor cells and cell clusters was collected. The filtrate was centrifuged at 1000 rpm at room temperature for 5 minutes, the supernatant was discarded, and the cell pellet was resuspended in 1 mL of DPBS and transferred to a 1.5 mL EP tube. The cell pellet was then rapidly centrifuged for 10–15 seconds using a handheld centrifuge. After repeating the above DPBS washing procedure once, the cell pellet was resuspended in 0.5–1 mL of erythrocyte lysis buffer. After standing at room temperature for 5 minutes, an appropriate amount of DMEM medium containing 1% fetal bovine serum was added to terminate the reaction. The cell pellet was then rapidly centrifuged, the supernatant was removed, and the cell pellet was washed twice with DPBS. Resuspend the cells in a suitable amount of matrix gel after washing (the entire cell resuscitation process is performed on ice). Seed 20 μL per well in a 24-well plate and incubate at 37°C for 15–20 minutes. After the matrix gel has completely solidified, add 0.5 mL of small cell lung cancer organoid culture medium (medium 1) to each well and continue culturing. Observe the cell digestion under a microscope. Figure 3 The medium is changed every 3 to 4 days during the culture process. After about 4 days of culture, small cell lung cancer organoid cell clusters can be seen. After one week of culture, the diameter of the primary small cell lung cancer organoid cell clusters is about 50 to 100 μm. Under these conditions, mouse Trp53 / Rb1 / Pten-deficient small cell lung cancer organoids can be obtained.

[0152] (3) Passaging method for organoids of mouse Trp53 / Rb1 / Pten-deficient small cell lung cancer. The primary small cell lung cancer organoids cultured above were observed under a microscope. When more than 80% of the organoids had a diameter greater than 50 μm and the organoid density was high, organoid passage could be performed. Remove the culture medium, resuspend the matrix gel in the well plate with 1 mL of pre-cooled DPBS, and transfer it to a 1.5 mL EP tube. Centrifuge rapidly for 10 seconds, discard the supernatant, and resuspend the organoid pellet in 1 mL of DPBS. Use a pipette to repeatedly pipette and blow 10-15 times to mechanically disperse the small cell lung cancer organoid cell clusters. Centrifuge rapidly for another 10 seconds, remove the supernatant, place the plate on ice, add an appropriate amount of matrix gel to resuspend the cell pellet, and re-seed it in 20 μL per well in a 24-well plate. Incubate at 37°C for 15-20 minutes until the matrix gel is completely solidified. Add 0.5 mL of small cell lung cancer organoid-specific culture medium (medium 1) to each well and continue culturing at 37°C in a 5% CO2 incubator. Change the medium every 3-4 days during the culture period and observe the growth of small cell lung cancer organoids regularly.

[0153] Example 4. The procedure for culturing small cell lung cancer organoids is basically the same as that in Example 3, except that the culture medium 2 prepared in Example 2 is a special culture medium for small cell lung cancer organoids.

[0154] Example 5. Morphological identification of organoids from Trp53 / Rb1 / Pten-deficient small cell lung cancer

[0155] After establishing Trp53 / Rb1 / Pten-deficient small cell lung cancer organoids, the morphology of the organoids cultured in Example 3 was identified. The growth of each generation of organoids was observed in real-time under a microscope. After 3 days of culture in the 0th generation, SCLC cells began to proliferate rapidly, forming round, vesicular, three-dimensional organoid tissue structures. Figure 4 Four days after passage 1, SCLC organoids showed some morphological differences, exhibiting a mixed morphology of solid and vesicular shapes, which to some extent conforms to the morphological characteristics of tumor organoids. Figure 5 It is worth noting that after 5 days of second-generation culture, the morphological differences of SCLC organoids were more significant, with some SCLC organoids exhibiting cell protrusion characteristics and displaying a phenotype with strong invasive capabilities. Figure 6 ), and also exist solid, budding, vesicular, and mixed morphologies of SCLC organoids ( Figure 7 This indicates strong cellular heterogeneity in SCLC organoids, resulting in significant morphological and phenotypic differences in the cultured organoids. Under specific culture medium conditions, these SCLC organoids maintained multiple morphologies even after multiple passages; for example, after the 5th passage, the SCLC organoids still exhibited solid, vesicular, and mixed morphologies. Figure 8Furthermore, the protruding, highly invasive SCLC organoids can also be well passaged and maintained. Figure 9 Morphological identification of the small cell lung cancer organoids cultured in Example 4 was performed, and the results were basically consistent with those in Example 3. This demonstrates that successful culture of Trp53 / Rb1 / Pten-deficient small cell lung cancer organoids can be achieved under the same culture medium composition.

[0156] Example 6. Pulmonary neuroendocrine cell markers and Trp53 / Rb1 / Pten gene knockout identification in organoids of Trp53 / Rb1 / Pten-deficient small cell lung cancer

[0157] The cultured SCLC organoids were further identified using RT-qPCR to determine the mRNA levels of relevant pulmonary neuroendocrine cell markers and the Trp53 / Rb1 / Pten genes. The original organoid culture medium in the 24-well plate was aspirated using a pipette, and 500 μL of DPBS solution was added. The gel droplets were pipetted to detach from the bottom of the culture dish, and the organoids and droplets were collected in 1.5 mL RNase-free EP tubes. The tubes were centrifuged at 500 g for 3 minutes at room temperature, the supernatant was discarded, and the cell pellet was washed once more with DPBS. After centrifugation, the cell pellet was dissolved in 1 mL of Trizol to extract organoid RNA, which was then used for RT-qPCR experiments. The results are shown below. Figure 10 As shown, neuroendocrine cell markers were highly expressed in SCLC organoids, and the mRNA level of the Trp53 / Rb1 / Pten gene was low. These results suggest that the Trp53 / Rb1 / Pten knockout efficiency is good in Trp53 / Rb1 / Pten-deficient small cell lung cancer organoids, and that SCLC organoids can maintain neuroendocrine cell characteristics well.

[0158] Example 7. Construction of an organoid microarray model for Trp53 / Rb1 / Pten-deficient small cell lung cancer

[0159] To construct a three-dimensional respiratory chip, either a respiratory microarray or the commercially available Emulate S1 chip was used. SCLC organoids cultured to passage 3 or higher were aspirated from the matrix gel in the microarray, transferred to 1.5 mL EP tubes, and centrifuged briefly for 10 seconds. The supernatant was removed, and the organoid pellet was resuspended in 1 mL of 1×DPBS. After another 10-second centrifugation, the DPBS was removed, and an appropriate amount of matrix gel was added to resuspend the SCLC organoids. The resuspended organoids were then rapidly perfused into the microarray channels, avoiding the formation of air bubbles. The microarray was incubated at 37°C for 15–20 minutes until the matrix gel solidified. Then, Trp53 / Rb1 / Pten-deficient small cell lung cancer organoid culture medium was perfused into the microarray, and the microarray was cultured in a cell culture incubator. When SCLC organoids grow to a suitable density within the microarray, the manipulability of the dynamic microenvironment within the respiratory organ-on-a-chip allows for further mechanical stretching to simulate the in vivo lung respiratory microenvironment. This enables the construction of a complete organoid microarray model for Trp53 / Rb1 / Pten-deficient small cell lung cancer, which better reflects the physiological correlation of Trp53 / Rb1 / Pten-deficient small cell lung cancer development. This provides a more reliable in vitro technology platform for exploring the relationship between the dynamic microenvironment and Trp53 / Rb1 / Pten-deficient small cell lung cancer cells and for drug screening.

[0160] Comparative Example

[0161] The procedure for culturing small cell lung cancer organoids was basically the same as in Example 3. Mouse tumor tissue was digested into single cells, and after lysing red blood cells, the cells were seeded into well plates. The culture medium specifically for small cell lung cancer organoids used was either medium 3 or medium 4 prepared in Example 2. On the fourth day of culture, the morphological identification of the cultured organoids was performed according to the method in Example 5. The results are as follows. Figure 11 As shown. From Figure 11 It can be seen that the lack of Noggin results in poor organoid condition, with most remaining as single cells or cell clusters, growing slowly, and not easily forming organoids; while organoids cultured without CHIR-99021 are smaller in size and have poorer cell viability.

[0162] Therefore, the culture medium of this invention, through the screening and addition of cytokine components, particularly the combined use of Noggin and CHIR-99021, can produce small cell lung cancer organoids with larger cell volume, better cell condition, shorter doubling time, more cells obtained in the same time period, and faster passage. Furthermore, the small cell lung cancer organoid culture medium provided by this invention can better maintain the invasiveness and heterogeneity of tumor cells during culture, and the genetic characteristics remain stable during organoid culture and passage. The technical solution of this invention is beneficial for the widespread application of small cell lung cancer organoids in experimental research and has significant application value.

[0163] The inventors obtained Trp53 / Rb1 / Pten gene knockout small cell lung cancer by knocking out the Trp53 / Rb1 / Pten gene in mice. Through screening and optimization of the main components of the small cell lung cancer organoid culture medium, especially the added major cytokine components, they successfully obtained Trp53 / Rb1 / Pten gene-deficient small cell lung cancer organoids. These organoids, under the culture conditions of this invention, better maintain the invasiveness and heterogeneity of tumor cells, and their gene characteristics remain stable during organoid culture passages.

[0164] In addition, this invention also obtained an organoid model and organoid chip of small cell lung cancer (Trp53 / Rb1 / Pten gene mutant), which simulates the characteristics of real SCLC in vitro and can be used for the detection of drug sensitivity in clinical oncology patients and the testing and application of potential effective target drugs.

[0165] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A culture medium for small cell lung cancer organoids, comprising a basal culture medium, an antimicrobial agent, a culture medium supplement, cytokines, and optionally a buffer.

2. The small cell lung cancer organoid culture medium according to claim 1, characterized in that, The small cell lung cancer is a Trp53 / Rb1 / Pten gene mutant small cell lung cancer, preferably a Trp53 / Rb1 / Pten gene deletion small cell lung cancer.

3. The small cell lung cancer organoid culture medium according to claim 1 or 2, characterized in that, The basal culture medium is selected from MEM, BME, RPMI-1640, DMEM, DMEM / F12 or Advanced DMEM / F12 culture medium, preferably DMEM / F12 or Advanced DMEM / F12 culture medium, and more preferably Advanced DMEM / F12 culture medium.

4. The small cell lung cancer organoid culture medium according to claim 1 or 2, characterized in that, The antimicrobial agent is selected from penicillin, streptomycin, aminoglycoside antibiotics, amphotericin B, ornidazole, primocin, or any combination thereof; preferably, the antimicrobial agent comprises penicillin, streptomycin, and primocin.

5. The small cell lung cancer organoid culture medium according to claim 1 or 2, characterized in that, The culture medium supplement comprises one or more of the following: fetal bovine serum, newborn calf serum, human serum, B27 supplement, N2 supplement, amino acids selected from L-glutamine, N-acetylcysteine, nicotinamide, GlutaMAX or any combination thereof, and ascorbic acid; preferably, the culture medium supplement comprises B27 supplement, N-acetylcysteine ​​and GlutaMAX.

6. The small cell lung cancer organoid culture medium according to claim 1 or 2, characterized in that, The cytokines are selected from WNT signaling pathway activators, bone morphogenetic protein inhibitors, epidermal growth factor, fibroblast growth factor, transforming growth factor, vascular endothelial growth factor, hepatocyte growth factor, bone morphogenetic protein, neuromodulatory protein, ROCK inhibitors, transforming growth factor β inhibitors, p38 MAPK inhibitors, MDM2 inhibitors, or any combination thereof; preferably, the cytokines comprise WNT signaling pathway activators, bone morphogenetic protein inhibitors, fibroblast growth factor, ROCK inhibitors, transforming growth factor β inhibitors, and p38 MAPK inhibitors.

7. The small cell lung cancer organoid culture medium according to claim 6, characterized in that, The WNT signaling pathway activator is selected from GSK3β inhibitors, R-spondin protein, Wnt protein, or any combination thereof. Preferably, the WNT signaling pathway activator is selected from a combination of GSK3β inhibitors and R-spondin protein; and / or The bone morphogenetic protein inhibitor is selected from Noggin, ML347, DMH1, LDN-212854, LDN-214117, LDN-193189, Dorsomorphin, osteosclerosing protein, or any combination thereof, preferably Noggin; and / or The fibroblast growth factor is selected from fibroblast growth factor 1 (FGF1), fibroblast growth factor 2 (FGF2), fibroblast growth factor 7 (FGF7), fibroblast growth factor 9 (FGF9), fibroblast growth factor 10 (FGF10), or any combination thereof, preferably FGF2; and / or The vascular endothelial growth factor is selected from vascular endothelial growth factor A, vascular endothelial growth factor B, vascular endothelial growth factor C, or any combination thereof; and / or The ROCK inhibitor is selected from Y-27632, Fasudi HCl, Thiazovivin, GSK42928A, ZINC00881524, RKI-1447, AT13148, Belumosudil, GSK269962A HCl, Chroman 1, or any combination thereof, preferably Y-27632; and / or The transforming growth factor β inhibitor is selected from RepSox, E-616452, A83-01, A77-01, SB431542, GW788388, TGFβRI-IN-2, BIBF0775, ITD1, SJN2511, D4476, LY364947, SB505124, SB525334, SD208, Activin A, or any combination thereof, preferably A83-01; and / or The p38 MAPK inhibitor is selected from SB203580, Doramapimod (BIRB796), SB202190, LY2228820, VX-702, PH-797804, VX-745, TAK-71599, BMS-582949, or any combination thereof, preferably SB202190; and / or The MDM2 inhibitor is selected from Rebemadlin, Idasanutlin, Nutlin-3, Nutlin-3a, Nutlin-3b, MX69, NVP-CGM097, MI-773, RG-7112, HDM201, YH239-EE, NSC207895, Serdemetan, or any combination thereof.

8. The small cell lung cancer organoid culture medium according to claim 7, characterized in that, The GSK3β inhibitor is selected from CHIR-99021, GSK3β inhibitor IX, GSK3β inhibitor VII, Indirubin, L803-mts, TWS119, AZD2858, AR-A014418, TDZD-8, LY2090314, 2-D08, IM-12, 1-Azakenpaullone, SB216763, LY2090314, CHIR-98014, BIO-acetoxime, SAR502250, trihydrochloride, or any combination thereof, preferably CHIR-99021; and / or The R-spondin protein is selected from R-spondin 1, R-spondin 2, R-spondin 3, R-spondin 4, or any combination thereof, preferably R-spondin 1; and / or The Wnt protein is selected from Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b, Wnt9a, Wnt9b, Wnt10a, Wnt10b, Wnt11, Wnt16, or any combination thereof.

9. The small cell lung cancer organoid culture medium according to claim 1 or 2, characterized in that, The buffer is selected from bicarbonate, HEPES, HEPPSO, MOPS, POPSO, PIPES, DIPSO, MES, TES, BES or any combination thereof, with HEPES being preferred.

10. The small cell lung cancer organoid culture medium according to claim 1 or 2, characterized in that, The small cell lung cancer organoid culture medium contains: The basal culture medium, wherein the basal culture medium is selected from Advanced DMEM / F12 medium; An antimicrobial agent, wherein the antimicrobial agent is selected from penicillin / streptomycin bispecific antibiotics, primocin, or a combination thereof; Culture medium supplement, wherein the culture medium supplement is selected from B27 supplement, N-acetylcysteine, GlutaMAX, or any combination thereof; Cytokines, wherein the cytokines are selected from Noggin, R-spondin 1, CHIR-99021, FGF2, Y-27632, A83-01, SB202190 or combinations thereof, preferably, the cytokines are composed of Noggin, R-spondin 1, CHIR-99021, FGF2, Y-27632, A83-01 and SB202190; And a buffer, wherein the buffer is selected from HEPES.

11. The small cell lung cancer organoid culture medium according to claim 9, characterized in that, The small cell lung cancer organoid culture medium contains: GlutaMAX, 0.5–10 mM; HEPES, 5–20 mM; penicillin / streptomycin antibiotics, 0.5–3%; Primocin, 10–80 ng / mL; Noggin, 50–200 ng / mL; B27 supplement, 0.5–10 mM; N-acetylcysteine, 0.1–5 mM; R-Spondin 1, 100–1000 ng / mL; CHIR-99021, 1–10 μM; FGF2, 2–10 ng / mL; Y-27632, 1–10 μM; A83-01, 500–2000 nM; SB202190, 1–10 μM.

12. A method for culturing organoids from small cell lung cancer, comprising the following steps: Add the organoid culture medium for small cell lung cancer as described in any one of claims 1-11 to a three-dimensional scaffold containing tumor cells and / or tumor cell clusters of small cell lung cancer.

13. The method for culturing small cell lung cancer organoids according to claim 12, characterized in that, The three-dimensional scaffold is selected from synthetic biological scaffolds, natural scaffolds, or any combination thereof.

14. The method for culturing small cell lung cancer organoids according to claim 13, characterized in that, The synthetic biological scaffold is selected from porous solids, nanofibers, or hydrogels.

15. The method for culturing small cell lung cancer organoids according to any one of claims 12-14, characterized in that, The small cell lung cancer mentioned above is a Trp53 / Rb1 / Pten gene mutant small cell lung cancer.

16. The method for culturing small cell lung cancer organoids according to any one of claims 12-15, characterized in that, The tumor cells and / or tumor cell clusters are obtained by a method comprising the following steps: contacting digestive fluid with small cell lung cancer tumor tissue.

17. The method for culturing small cell lung cancer organoids according to claim 16, characterized in that, The digestive fluid is lung tissue digestive fluid, which contains one or more of the following: collagenase, calcium chloride, zinc chloride, DNase, neutral protease, fetal bovine serum, Liberase enzyme, and antibiotics. Preferably, the DNAase is DNAase I; and / or The collagenase is selected from collagenase I, collagenase II, collagenase III, collagenase IV, collagenase V, or any combination thereof, preferably collagenase I; and / or The antibiotic in question is a penicillin / streptomycin bispecific antibody.

18. The method for culturing small cell lung cancer tumor organoids according to claim 17, characterized in that, The lung tissue digestion fluid contains collagenase I, DNAase I, and penicillin / streptomycin antibiotics; preferably, the concentration of collagenase I is in the range of 0.1 mg / mL to 5 mg / mL, more preferably 1 mg / mL; the concentration of DNAase I is in the range of 0.1 to 0.5 mg / mL; and the concentration of penicillin / streptomycin antibiotics is in the range of 0.5% to 3%, more preferably 1%.

19. The method for culturing small cell lung cancer organoids according to any one of claims 12-18, characterized in that, The culture method also includes changing the medium every 3 to 4 days, and passage of organoids when the organoid density of small cell lung cancer reaches 80%.

20. A small cell lung cancer organoid obtained by the culture method according to any one of claims 12-19.

21. An organoid for small cell lung cancer with a Trp53 / Rb1 / Pten gene mutation.

22. The Trp53 / Rb1 / Pten gene mutant small cell lung cancer organoid according to claim 21, obtained by the culture method according to any one of claims 12-19.

23. Use of small cell lung cancer organoids according to any one of claims 20-22, including toxicity testing of the drug, discovery, screening and validation of new drugs.

24. A small cell lung cancer organoid chip, comprising the small cell lung cancer organoid as described in any one of claims 20-22.

25. The organoid chip for small cell lung cancer according to claim 24, characterized in that, The small cell lung cancer organoid chip is a three-dimensional respiratory chip that can simulate the physiological microenvironment of lung respiration in vivo.

26. The organoid chip for small cell lung cancer according to claim 25, characterized in that, The material of the respiratory three-dimensional chip is selected from polydimethylsiloxane, glass, silicon, plexiglass, paper, polycaprolactone, polyurethane, or any combination thereof.

27. The small cell lung cancer organoid microarray according to any one of claims 24-26, characterized in that, The breathing 3D chip is prepared by a method selected from the following: photolithography, molding, micro-contact imprinting, laser etching, machining, and 3D printing.

28. A method for screening targeted drugs for small cell lung cancer, comprising: Contact the small cell lung cancer organoids of any one of claims 20-22 or the small cell lung cancer organoid microarrays of any one of claims 24-27 with the candidate reagents.

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