A culture medium for oral cancer organoid culture, and a culture method and application thereof

Abstract

The present application relates to a culture medium for oral cancer organoid culture, comprising an MST1 / 2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, a fibroblast growth factor, CHIR99021, an epidermal cell growth factor, insulin, an ITS cell culture additive, SB202190, Y27632, dexamethasone, GlutaMAX, and non-essential amino acids. The present application also relates to a culture method of oral cancer organoids and the use thereof. By using the oral cancer organoid culture medium of the present application, the effective and rapid expansion of oral cancer organoids can be achieved, the organoids obtained by expansion retain the pathological characteristics of the patient, the culture success rate and expansion rate of the oral cancer organoids are improved, and a research basis can be provided for personalized treatment of the patient.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a culture medium for culturing oral cancer organoids, a method for culturing oral cancer organoids using the culture medium, and its application in drug efficacy evaluation and screening. Background Technology

[0002] Oral cancer is a general term for malignant tumors occurring in the oral cavity. It includes cancers originating in the tongue (anterior two-thirds of the tongue), buccal mucosa, gingiva, floor of the mouth, and hard palate. It is one of the most common malignant tumors in the head and neck region, ranking sixth globally, after nasopharyngeal carcinoma. Oral cancer can occur at any age, with a peak incidence between 40 and 60 years of age. The male-to-female ratio is approximately 2:1. In recent years, oral cancer has shown the following new characteristics: First, the incidence of tongue cancer is increasing rapidly, now accounting for nearly half of all oral cancers; second, the age of onset is trending younger, with many patients aged 20-30; and third, the number of female patients is increasing year by year. Over the past 20 years, although traditional treatment methods, including surgery, radiotherapy, and chemotherapy, have made varying degrees of progress, the prognosis of oral cancer remains unsatisfactory, with a 5-year survival rate of only about 50% to 60%. Approximately one-third of patients experience recurrence, and surgical treatment severely impacts swallowing, speech function, and facial appearance, resulting in significant and severe disfigurement, eating, and speech difficulties. The main reasons affecting survival are local recurrence and lymphatic metastasis. Therefore, new treatment methods, such as targeted tumor therapy and other biological therapies, are receiving increasing attention. Simultaneously, improving the anti-cancer efficacy of drug therapy and reducing drug toxicity and side effects—that is, the targeted nature of anti-cancer treatment—is a key concern in basic and clinical research on tumors.

[0003] Traditional clinical drug sensitivity testing mostly employs two-dimensional cell culture. However, two-dimensional cultured cells only simulate tissue physiological conditions to a limited extent, lacking the real tissue structure in vivo, easily leading to low differentiation levels and loss of cellular physiological functions, thus making it difficult to predict actual clinical outcomes. Organoids, belonging to three-dimensional (3D) cell cultures, are mainly derived from human embryonic stem cells, induced pluripotent stem cells, and adult stem cells with differentiation capacity. Endogenous tissue stem cells exist in different tissues and organs, playing a crucial role in maintaining the functional morphology of each organ. Under certain in vitro induction conditions, these stem cells can self-organize into miniature structures with a diameter of only a few millimeters. Tumor organoids are miniature 3D tumor cell models cultured in the laboratory using primary tumors taken from patients. Tumor organoids highly simulate the characteristics of the source tumor tissue, preserving tumor heterogeneity between individuals, and can be used for functional testing, such as high-throughput drug screening and personalized precision treatment.

[0004] Currently, oral cancer organoid culture methods mostly employ basal media (DMEM or DMEM / F12), R-spondin-1, Noggin, and some expensive protein factors, resulting in high organoid culture costs. Furthermore, the complexity and technical difficulty of this technology limit its large-scale commercial application. Therefore, there is a need to develop a low-cost, simple, and highly successful organoid culture method and culture medium. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a culture medium and a culture method for rapidly expanding oral cancer organoids in vitro.

[0006] One aspect of the present invention is to provide a culture medium for oral cancer organoids, the culture medium comprising an MST1 / 2 kinase inhibitor, at least one cell culture additive selected from N2 and B27, fibroblast growth factor, CHIR99021, epidermal growth factor, insulin, ITS cell culture additive, SB202190, Y27632, dexamethasone, GlutaMAX, and non-essential amino acids. The MST1 / 2 kinase inhibitor comprises a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof.

[0007]

[0008] in,

[0009] R1 is selected from C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C2-C6 spirocycloalkyl, and aryl (e.g., phenyl and naphthyl), aryl C1-C6 alkyl (e.g., benzyl), and heteroaryl (e.g., thiophene).

[0010] R2 and R3 are each independently selected from C1-C6 alkyl groups, preferably C1-C3 alkyl groups, and more preferably methyl groups;

[0011] R4 and R5 are each independently selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, C4-C8 cycloalkylalkyl, C1-C6 alkylhydroxy, C1-C6 haloalkyl, C1-C6 alkylaminoC1-C6 alkyl, C1-C6 alkoxyC1-C6 alkyl, and C3-C6 heterocyclicC1-C6 alkyl (the heterocyclic group is selected from, for example, piperidinyl, tetrahydropyranyl, etc.);

[0012] R6 is selected from halogens (preferably fluorine and chlorine, more preferably fluorine), C1-C6 alkyl (preferably methyl), C1-C6 alkoxy (preferably methoxy), and C1-C6 haloalkyl (preferably trifluoromethyl).

[0013] In a preferred embodiment, the MST1 / 2 kinase inhibitor comprises a compound of formula (Ia) or a pharmaceutically acceptable salt or solvate thereof.

[0014]

[0015] in,

[0016] R1 is selected from C1-C6 alkyl, phenyl optionally substituted with 1-2 independent R6, thiophene group optionally substituted with 1-2 independent R6, and benzyl optionally substituted with 1-2 independent R6, more preferably phenyl optionally substituted with 1-2 independent R6.

[0017] R5 is selected from hydrogen, C1-C6 alkyl, and C3-C6 cycloalkyl, with hydrogen being more preferred;

[0018] R6 is independently selected from halogens, C1-C6 alkyl groups, and C1-C6 haloalkyl groups, with R6 more preferably being fluorine, methyl, or trifluoromethyl.

[0019] Preferably, the MST1 / 2 inhibitor is at least one selected from the following compounds or their pharmaceutically acceptable salts or solvates.

[0020]

[0021]

[0022]

[0023]

[0024]

[0025] Most preferably, the MST1 / 2 kinase inhibitor of the present invention is compound 1.

[0026] In embodiments of the present invention, the content of each component in the culture medium of the present invention satisfies any one or more, or all of the following:

[0027] (1) The concentration of the MST1 / 2 kinase inhibitor is preferably 2.5–10 μM;

[0028] (2) The volume ratio of B27 or N2 cell culture additive to culture medium is 1:25 to 1:100;

[0029] (3) The preferred concentration of fibroblast growth factor is 3 to 30 ng / mL;

[0030] (4) The preferred concentration of CHIR99021 is 1.25–5 μM;

[0031] (5) The preferred concentration of epidermal growth factor is 2–18 μM;

[0032] (6) The preferred insulin concentration is 1–10 μg / mL;

[0033] (7) The volume ratio of ITS cell culture additive to culture medium is preferably 1:30 to 1:300;

[0034] (8) The preferred concentration of SB202190 is 100–400 nM;

[0035] (9) The preferred concentration of Y27632 is 3–30 μM;

[0036] (10) The concentration of dexamethasone is preferably 0.02–0.5 μM;

[0037] (11) The volume ratio of GlutaMAX to the culture medium is preferably 1:30 to 1:300;

[0038] (12) The non-essential amino acids are selected from one or more of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine, and the concentration of the non-essential amino acids is preferably 50 to 200 μM.

[0039] In embodiments of the present invention, the culture medium further contains an initial culture medium selected from DMEM / F12, DMEM, F12 or RPMI-1640; and one or more antibiotics selected from streptomycin / penicillin, amphotericin B and Primocin.

[0040] In a preferred embodiment, when the antibiotic is selected from streptomycin / penicillin, the concentration range of streptomycin is 25-400 μg / mL, the concentration range of penicillin is 25-400 U / mL, when the antibiotic is selected from amphotericin B, the concentration range is 0.25-4 μg / mL, and when the antibiotic is selected from Primocin, the concentration range is 25-400 μg / mL.

[0041] The present invention also provides a method for culturing oral cancer organoids. In the method for culturing oral cancer organoids of the present invention, the oral cancer organoids are cultured using the oral cancer organoid culture medium of the present invention.

[0042] The oral cancer organoid culture method of the present invention includes the following steps.

[0043] 1. Isolate samples from oral cancer solid tumor tissue to obtain primary oral cancer cells. This process includes the following steps:

[0044] (1) Separate oral cancer tissue samples, add basal culture medium and tissue digestion solution in a 1:3 ratio (the amount of tissue digestion solution added is about 10 mL of tissue digestion solution per 1 g of tumor tissue) and place them in a constant temperature shaker for digestion. The digestion temperature is 4 to 37°C, the shaker speed is 200 rpm to 350 rpm, and the digestion time is 3 to 6 hours.

[0045] (2) After digestion, centrifuge and discard the supernatant. The centrifugation speed is 1200-1600 rpm and the centrifugation time is 2-6 minutes.

[0046] The basal culture medium formulation includes an initial medium selected from DMEM / F12, DMEM, F12, or RPMI-1640; and one or more antibiotics selected from streptomycin / penicillin, amphotericin B, and Primocin. The tissue digestion solution formulation includes RPMI-1640 medium, collagenase II (1–2 mg / mL), collagenase IV (1–2 mg / mL), DNase (50–100 U / mL), hyaluronidase (0.5–1 mg / mL), calcium chloride (1–5 mM), and bovine serum albumin (BSA) (5–10 mg / mL).

[0047] 2. Prepare the oral cancer organoid culture medium of the present invention, and culture the primary oral cancer cells obtained in the above steps.

[0048] The primary oral cancer cells obtained in step 1 above were resuspended in the oral cancer organoid culture medium of the present invention and counted, and the cell density was diluted to 5-10 × 10⁻⁶. 6 Cells / mL, take out the diluted cell suspension and add it to an equal volume of Matrigel and mix well. Then, seed the mixture into a multi-well plate and place the seeded multi-well plate in an incubator for 30-60 minutes until Matrigel is completely solidified. Then, add oral cancer organoid culture medium for expansion culture.

[0049] The present invention also provides a method for evaluating or screening drugs for treating oral cancer, comprising the following steps:

[0050] (1) Oral cancer organoids are cultured using the oral cancer organoid culture method of the present invention;

[0051] (2) Select the drug to be tested and dilute it according to the required concentration gradient;

[0052] (3) Add the diluted drug to the organoids cultured in (1);

[0053] (4) Perform organoid size or organoid viability tests.

[0054] The beneficial effects of this invention include:

[0055] (1) Improve the success rate of oral cancer organoid culture, with a success rate of over 90%;

[0056] (2) Ensure that the oral cancer organoids cultured in vitro can maintain the pathological characteristics of the patients;

[0057] (3) It has high amplification efficiency and can quickly culture oral cancer organoids. The amplified oral cancer organoids can also be continuously passaged.

[0058] (4) The culture cost is controllable, and the culture medium does not need to be added with expensive Wnt agonists, R-spondin family proteins, Noggin protein, BMP inhibitors, fibroblast growth factor 10 (FGF10) and other factors.

[0059] (5) The oral cancer organoids obtained by the technology are large in number, which is suitable for high-throughput screening of candidate compounds and providing patients with high-throughput in vitro drug sensitivity functional tests. Attached Figure Description

[0060] Figure 1A-1L The figure illustrates the effect of different concentrations of factors added to the oral cancer organoid culture medium of the present invention on the proliferation of oral cancer organoids.

[0061] Figure 2A-2D To observe under a microscope photographs of oral cancer organoids cultured using the oral cancer organoid culture medium of the present invention, wherein... Figure 2A This shows a photograph of the organoid obtained from sample OZ-018 after 8 days of culture; Figure 2B This shows a photograph of the organoid obtained from sample OZ-019 10 days later; Figure 2C This shows a photograph of the organoid obtained from sample OZ-020 after 7 days of culture; Figure 2D This shows a photograph of the organoid obtained from sample OZ-021 after 8 days of culture.

[0062] Figure 3A The results of pathological and immunohistochemical identification of oral cancer organoids obtained from sample OZ-020 using the oral cancer organoid culture medium of the present invention; Figure 3B The results of pathological and immunohistochemical identification of sample OZ-020 tissue; Figure 3C The results of pathological and immunohistochemical identification of oral cancer organoids obtained from sample OZ-021 using the oral cancer organoid culture medium of the present invention; Figure 3D The results of pathological and immunohistochemical identification of sample OZ-021 tissue are shown.

[0063] Figure 4 shows a comparison of the oral cancer organoid culture medium of the present invention with that of existing culture media for culturing oral cancer organoids, wherein... Figure 4AThe image shows a photograph taken after 10 days of culture in the OC-3 medium of the present invention. Figure 4B This shows a photograph taken after 10 days of culture in ED medium. Figure 4C This is a bar chart showing the relative sizes of organoids cultured in OC-3 and ED media.

[0064] Figure 5 shows the results of different drug sensitivity tests on oral cancer organoids cultured using the oral cancer organoid culture medium of the present invention. Figure 5A The images show organoid growth before drug treatment and organoid growth 3 days after drug treatment. Figure 5B The bar chart shows the inhibition rate of different concentrations of the test drug on the growth of oral cancer organoids. Detailed Implementation

[0065] To better understand the present invention, it will be further described below with reference to embodiments and accompanying drawings. The following embodiments are merely illustrative and not intended to limit the scope of the invention.

[0066] [Preparation Examples of MST1 / 2 Kinase Inhibitors]

[0067] In this specification, an MST1 / 2 kinase inhibitor refers to any inhibitor that directly or indirectly negatively regulates MST1 / 2 signaling. Generally, an MST1 / 2 kinase inhibitor binds to MST1 / 2 kinases and reduces their activity, for example. Due to the structural similarity between MST1 and MST2, an MST1 / 2 kinase inhibitor can also be a compound that binds to either MST1 or MST2 and reduces their activity, for example.

[0068] 1. Preparation of MST1 / 2 kinase inhibitor compound 1: 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzene sulfonamide 1

[0069]

[0070] Methyl 2-amino-2-(2,6-difluorophenyl)acetate (A2): In a round-bottom flask, 2.0 g of 2-amino-2-(2,6-difluorophenyl)acetic acid was added, followed by 30 mL of methanol, and then 1.2 mL of thionyl chloride was added dropwise under ice bath conditions. The reaction mixture was allowed to react overnight at 85 °C. After the reaction was complete, the solvent was evaporated under reduced pressure, yielding a white solid, which was used directly in the next step.

[0071] 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetic acid methyl ester (A3): 2 g of 2-amino-2-(2,6-difluorophenyl)acetic acid methyl ester was added to a round-bottom flask, followed by 30 mL of acetone and 2.2 g of potassium carbonate. The system was then cooled to -10 °C using an ice-salt bath. A solution of 3.1 g of 2,4-dichloro-5-nitropyrimidin in acetone was then slowly added. The reaction mixture was stirred overnight at room temperature. After the reaction was complete, the mixture was filtered, and the solvent was removed from the filtrate under reduced pressure. The residue was purified by pressurized silica gel column chromatography to give compound A3. LC / MS: M+H 359.0.

[0072] 2-Chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridine-6(5H)-one (A4): Methyl 2-((2-chloro-5-nitropyrimidin-4-yl)amino)-2-(2,6-difluorophenyl)acetate (2.5 g) was added to a round-bottom flask, followed by acetic acid (50 mL) and iron powder (3.9 g). The reaction system was stirred at 60 °C for two hours. After the reaction was completed, the solvent was evaporated under reduced pressure, and the product was neutralized to alkaline with saturated sodium bicarbonate. Extraction with ethyl acetate was performed, and the organic phase was washed with water and saturated brine, respectively, and dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated under reduced pressure to obtain the crude product. The crude product was washed with diethyl ether to give compound A4. LC / MS: M+H 297.0.

[0073] 2-Chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridine-6(5H)-one (A5): 2 g of 2-chloro-7-(2,6-difluorophenyl)-7,8-dihydropteridine-6(5H)-one and 10 mL of N,N-dimethylacetamide were added to a round-bottom flask. The mixture was cooled to -35 °C, and 0.9 mL of iodomethane was added, followed by sodium hydride (615 mg). The reaction mixture was stirred for two hours. After the reaction was complete, the mixture was quenched with water, extracted with ethyl acetate, and the organic phase was washed with water and saturated brine, respectively, and dried over anhydrous sodium sulfate. The organic phase was filtered and evaporated under reduced pressure to obtain the crude product. The crude product was washed with diethyl ether to obtain compound A5. LC / MS: M+H 325.0.

[0074] 4-((7-(2,6-difluorophenyl)-5,8-dimethyl-6-oxo-5,6,7,8-tetrahydropteridin-2-yl)amino)benzenesulfonamide (1): 2-chloro-7-(2,6-difluorophenyl)-5,8-dimethyl-7,8-dihydropteridin-6(5H)-one (100 mg), sulfonamide (53 mg), p-toluenesulfonic acid (53 mg), and sec-butanol (5 mL) were added to a round-bottom flask. The reaction mixture was stirred overnight at 120 °C. After the reaction was complete, the mixture was filtered and washed with methanol and diethyl ether to give compound 1. LC / MS: M+H 461.1.

[0075] 2. Preparation of other MST1 / 2 inhibitor compounds of the present invention

[0076] Other MST1 / 2 inhibitor compounds of the present invention were synthesized in a similar manner to compound 1, and their structures and mass spectrometry data are shown in the table below.

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083] Example 1: Effects of various added factors in oral cancer organoid culture medium on the proliferation of oral cancer organoids

[0084] (1) Preparation of oral cancer organoid culture medium

[0085] First, prepare the basal medium containing the initial culture medium. The initial culture medium can be selected from commonly used media in the art, such as DMEM / F12, DMEM, F12, or RPMI-1640. In this embodiment, the basal medium formulation is: DMEM / F12 medium (purchased from Corning) + 100 μg / mL Primocin (purchased from InvivoGen, 0.2% (v / v), commercially available product concentration 50 mg / mL).

[0086] Different types of additives (see Table 1) were added to the basal culture medium to prepare oral cancer organoid culture media containing different additives.

[0087] (2) Isolation and processing of primary oral cancer cells

[0088] 1 Sample Selection

[0089] Oral cancer solid tumor tissue samples (intraoperatively) were obtained from patients by professional medical personnel from a specialized medical institution, and all patients signed informed consent forms. The intraoperative sample size was 0.25cm. 3 The tissue was stored and transported using commercially available tissue preservation solution (manufacturer: Miltenyi Biotec).

[0090] 2. Material Preparation

[0091] After surface sterilization, 15mL sterile centrifuge tubes, pipettes, 10mL pipettes, and sterile pipette tips were placed in a laminar flow hood and irradiated with ultraviolet light for 30 minutes. The basal culture medium DMEM / F12 (Corning) was removed from the 4℃ freezer 30 minutes in advance, and the tissue digestion solution was removed from the -20℃ freezer 30 minutes in advance.

[0092] Tissue digestion solution formulation: 1640 medium (Corning, 10-040-CVR), collagenase II (2 mg / mL), collagenase IV (2 mg / mL), DNase (50 U / mL), hyaluronidase (0.75 mg / mL), calcium chloride (3.3 mM), bovine serum albumin (BSA) (10 mg / mL).

[0093] Collagenase II, collagenase IV, DNase, and hyaluronidase mentioned above were all purchased from Sigma-Aldrich; calcium chloride was purchased from Sangon Biotech (Shanghai) Co., Ltd.; and BSA was purchased from Biofroxx.

[0094] 3 Sample separation

[0095] 3.1 Tissue samples were taken from a laminar flow hood and placed in a culture dish. Blood-contaminated tissue was removed, and the samples were rinsed twice with basal culture medium. The tissue was then transferred to another culture dish and mechanically separated using a sterile scalpel, dividing the tissue blocks into 1*1*1mm pieces. 3 size;

[0096] 3.2 Transfer the dissected intraoperative tissue into a 15mL centrifuge tube, add 5mL of basal culture medium, mix well, and centrifuge at 1500rpm for 4 minutes;

[0097] 3.3 Discard the supernatant, add basal culture medium and tissue digestion solution in a 1:3 ratio (Note: the amount of tissue digestion solution added is about 10 mL of tissue digestion solution for 1 g of tumor tissue), label the sample name and number, seal with sealing film, and digest at 37°C and 300 rpm on a shaker (ZQLY-180N instrument). During the process, observe whether digestion is complete every 30 minutes. The judgment is based on the absence of visible particles.

[0098] 3.4 After digestion, the undigested tissue clumps were filtered out through a 100μm filter. The tissue clumps on the filter were rinsed with basal culture medium into centrifuge tubes to reduce cell loss. The tubes were then centrifuged at 1500rpm for 4 minutes at 25°C.

[0099] 3.5 Discard the supernatant and observe whether there are blood cells. If there are blood cells, add 8 mL of blood cell lysis buffer (purchased from Sigma), mix well, lyse at 4°C for 20 minutes, inverting and mixing once during the process, and centrifuge at 1500 rpm for 4 minutes at 25°C.

[0100] 3.6 Discard the supernatant, add 2 mL of basal culture medium to resuspend the cells, and set aside for later use.

[0101] 4. Cell counting and processing

[0102] 4.1 Microscopic observation: A small amount of resuspended cells were transferred and spread evenly in a culture dish. The density and morphology of cancer cells were observed under a microscope (CNOPTEC, BDS400).

[0103] 4.2 Viable cell counting: Take 12 μL of the resuspended cell suspension and 12 μL of trypan blue staining solution (manufacturer: Sangon Biotech (Shanghai) Co., Ltd.), mix thoroughly, and add 20 μL to a cell counting plate (manufacturer: Countstar, specification: 50 plates / box). Calculate the percentage of viable large cells (cell diameter >10 μm) using a cell counter (Countstar, IC1000) = viable cell count / total cell count * 100%.

[0104] (3) Culture of oral cancer organoids

[0105] The primary oral cancer cells obtained in the above steps were resuspended in pre-cooled DMEM / F12 and counted, with the cell density diluted to 5–10 × 10⁻⁶. 6 Cells / mL: 400 μL of diluted cell suspension was added to an equal volume of Matrigel (Corning) and gently mixed. The mixture was then seeded at 5 μL / well in a 96-well plate. The seeded plates were incubated for 30 minutes until the Matrigel completely solidified. Then, culture media (as shown in Table 1) that had been brought to room temperature were added, and the medium was changed every three days for expansion culture. After 7 days, the cultured organoids were photographed, and their diameters were measured and statistically analyzed to compare the promoting effects of various factors on the proliferation of oral cancer organoids. As a control, basal culture medium without any additives was used. The experimental results are shown in Table 1.

[0106] Table 1. Additives in the culture medium and their effects on promoting organoid proliferation.

[0107] 1 N2 Gibco 1:50 + 2 EGF R&D 5ng / mL + 3 R-spondin1 R&D 20ng / mL + 4 Prostaglandin E2 Tocris 0.5μM ○ 5 insulin Peprotech 1.5 μg / mL + 6 B27 Gibco 1:50 + 7 A8301 MCE 100nM - 8 SB202190 MCE 200nM + 9 bFGF R&D 10 ng / mL + 10 Hydrocortisone Sigma 10 ng / mL ○ 11 Noggin R&D 30ng / mL ○ 12 FBS Excell 5％ + 13 IGF-1 R&D 45ng / mL ○ 14 KGF R&D 5ng / mL ○ 15 GlutaMAX Gibco 1:100 + 16 Non-essential amino acids Corning 100μM + 17 Dexamethasone MCE 0.1μM + 18 NRG1 Sino biological 5ng / mL ○ 19 Y27632 MCE 10μM + 20 ITS Gibco 1:100 + 21 Compound 1 Preparation Example 5μM + 22 CHIR99021 MCE 2.5μM +

[0108] In this context, "+" indicates that, compared to the basal culture medium, the culture medium containing this additive promoted the proliferation of at least two oral cancer organoids isolated from oral cancer tissue; "-" indicates that, the culture medium containing this additive showed an inhibitory effect on the proliferation of at least one oral cancer organoid isolated from oral cancer tissue; and "○" indicates that, the culture medium containing this additive had no significant effect on the proliferation of at least two oral cancer organoids isolated from oral cancer tissue.

[0109] Based on the above results, we plan to select B27, fibroblast growth factor bFGF, CHIR99021, epidermal growth factor EGF, insulin, ITS cell culture additive, SB202190, Y27632, dexamethasone, GlutaMAX, compound 1, and non-essential amino acids for further culture experiments.

[0110] Example 2: Effects of different concentrations of added factors on the proliferation of oral cancer organoids

[0111] Oral cancer primary cells were obtained from intraoperative tissue samples (numbered OZ013 and OZ014) according to the method in Example 1(2), and organoid culture was performed using the methods in Table 2 below.

[0112] Table 2. Culture medium components (concentrations are final concentrations)

[0113]

[0114] When using Formula 1 culture medium, 200 μL of prepared B27 was added to each well of a 96-well plate inoculated with organoids, resulting in final concentrations of 1:25, 1:50, and 1:100 for B27. Control wells (BC) were also set up using Formula 1 culture medium. The final concentrations of other added factors in this series of media were the same as those in OC-3 medium. Experiments using Formulas 1-12 were conducted in the same manner and will not be described further.

[0115] When using the culture medium of Formula 2, 200 μL of the prepared bFGF was added to each well of a 96-well plate inoculated with organoids, with final concentrations of bFGF of 3 ng / mL, 10 ng / mL, and 30 ng / mL, respectively; and control wells (BC) were set up using the culture medium of Formula 2.

[0116] When using the culture medium of Formula 3, add 200 μL of prepared CHIR99021 to each well of a 96-well plate inoculated with organoids, based on Formula 3, with final concentrations of CHIR99021 of 1.25 μM, 2.5 μM, and 5 μM, respectively; and set up control wells (BC) using the culture medium of Formula 3.

[0117] When using the culture medium of Formula 4, 200 μL of the prepared EGF was added to each well of a 96-well plate inoculated with organoids, with final EGF concentrations of 2 ng / mL, 6 ng / mL, and 18 ng / mL, respectively; and control wells (BC) were set up using the culture medium of Formula 4.

[0118] When using the culture medium of Formula 5, 200 μL of prepared insulin was added to each well of a 96-well plate inoculated with organoids, with final insulin concentrations of 1 μg / mL, 3 μg / mL, and 10 μg / mL, respectively; and control wells (BC) were set up using the culture medium of Formula 5.

[0119] When using the culture medium of Formula 6, 200 μL of the prepared ITS was added to each well of a 96-well plate inoculated with organoids, with final ITS concentrations of 1:300, 1:100, and 1:30, respectively; and control wells (BC) were set up using the culture medium of Formula 6.

[0120] When using the culture medium of Formula 7, add 200 μL of the prepared SB202190 to each well of the 96-well plate inoculated with organoids, based on Formula 7, with final concentrations of SB202190 of 100 nM, 200 nM, and 400 nM, respectively; and set up control wells (BC) using the culture medium of Formula 7.

[0121] When using the culture medium of Formula 8, add 200 μL of prepared Y27632 to each well of the 96-well plate inoculated with organoids, based on Formula 8, with final concentrations of Y27632 of 3 μM, 10 μM and 30 μM, respectively; and set up control wells (BC) using the culture medium of Formula 8.

[0122] When using the culture medium of Formula 9, 200 μL of prepared dexamethasone was added to each well of a 96-well plate inoculated with organoids, with final concentrations of dexamethasone of 0.02 μM, 0.1 μM, and 0.5 μM, respectively; and control wells (BC) were set up using the culture medium of Formula 9.

[0123] When using the culture medium of Formula 10, 200 μL of prepared GlutaMAX was added to each well of a 96-well plate inoculated with organoids, with final concentrations of GlutaMAX of 1:300, 1:100, and 1:30, respectively; and control wells (BC) were set up using the culture medium of Formula 10.

[0124] When using the culture medium of Formula 11, 200 μL of the prepared compound 1 was added to each well of a 96-well plate inoculated with organoids, with final concentrations of 2.5 μM, 5 μM, and 10 μM, respectively; and control wells (BC) were set up using the culture medium of Formula 11.

[0125] When using the culture medium of Formula 12, 200 μL of the prepared non-essential amino acids were added to each well of a 96-well plate inoculated with organoids, with final concentrations of 50 μM, 100 μM, and 200 μM for the non-essential amino acids, respectively; and control wells (BC) were set up using the culture medium of Formula 12.

[0126] Seven days later, the cultured organoids were photographed, and their diameters were measured and statistically analyzed to compare the promoting effects of various factor concentrations on the proliferation of oral cancer organoids. Data from two samples are summarized below. Figures 1A-1L . Figures 1A-1L In this context, the ratio is the ratio of the diameter of organoids obtained after 7 days of culture in each culture medium to the diameter of the corresponding BC control wells after 7 days of culture. A ratio greater than 1 indicates that the culture medium containing different concentrations of factors or small molecule compounds promotes proliferation more effectively than the control well culture medium; a ratio less than 1 indicates that the culture medium containing different concentrations of factors or small molecule compounds promotes proliferation less effectively than the control well culture medium.

[0127] according to Figures 1A-1L As a result, the preferred volume concentration of B27 is 1:25 to 1:100; the preferred content of fibroblast growth factor bFGF is 3 to 30 ng / mL; the preferred content of CHIR99021 is 1.25 to 5 μM; the preferred content of epidermal growth factor EGF is 2 to 18 ng / mL; the preferred content of insulin is 1 to 10 μg / mL; the preferred volume concentration of ITS cell culture additive is 1:30 to 1:300; the preferred content of SB202190 is 100 to 400 nM; the preferred content of Y27632 is 3 to 30 μM; the preferred content of dexamethasone is 0.02 to 0.5 μM; the preferred volume concentration of GlutaMAX is 1:30 to 1:300; the preferred content of MST1 / 2 kinase inhibitor is 2.5 to 10 μM; and the preferred content of non-essential amino acids is 50 to 200 μM.

[0128] Example 3: Oral Cancer Organoid Culture and Identification

[0129] Oral cancer primary cells obtained according to the method described in Example 1(2) were resuspended in the oral cancer organoid culture medium OC-3 of the present invention and counted, and the cell density was diluted to 5-10 × 10⁻⁶. 6Cells / mL: Take 400 μL of the diluted cell suspension and add it to an equal volume of Matrigel (Corning). Mix gently, then seed the mixture at 50 μL / well in a 24-well plate. Place the seeded plate in an incubator for 30 minutes until the Matrigel is completely solidified. Then add 500 μL of pre-stored oral cancer organoid culture medium OC-3 to each well. Scale up the culture by changing the medium every three days.

[0130] On days 7-10, the cultured oral cancer organoids were observed using a microscope (Invitrogen EVOS M500). Figure 2A-2D These are photographs of oral cancer organoids obtained after culturing samples OZ-018, OZ-019, OZ-020, and OZ-021, taken under a 10x objective lens. Under the microscope, the oral cancer organoids appear as regular spherical shapes with relatively smooth surfaces.

[0131] The cultured oral cancer organoids were subjected to pathological and immunohistochemical identification, and the corresponding tissue samples were sent for pathological and immunohistochemical identification to compare the consistency between the organoid and tissue results.

[0132] Figure 3A and 3C The images show the pathological and immunohistochemical identification results of oral cancer organoids obtained from in vitro culture of samples OZ-020 and OZ-021, respectively, and are taken under a 20x objective lens. Figure 3A and 3C As shown, the HE results indicate that the organoid's structural morphology is that of cancerous tissue; the expression of CK, 34βE12, P63, CK5 / 6, and Ki67 suggests that the sample is oral cancer. Figure 3B and Figure 3D The pathological and immunohistochemical results of the tissues corresponding to OZ-020 and OZ-021 show that the oral cancer organoids cultured using the OC-3 culture medium of the present invention are consistent with the diagnostic results of oral cancer tissues.

[0133] Example 4: Comparison of culture effects with existing culture media

[0134] (1) Preparation of control culture medium

[0135] The culture medium used in the literature (Else Driehuis et al., Cancer Discov. 2019, 9:852-871) was formulated as follows: DMEM / F12 medium (Corning) + 1:50 B27 (Gibco) + 1.25 mmol / L N-acetylcysteine ​​(MCE) + 10 mmol / L nicotinamide (MCE) + 250 ng / ml amphotericin B (Selleck) + 10 μg / ml gentamicin (MCE) + 500 nM A8301 (MCE) + 50 ng / ml epidermal growth factor (R&D) + 10 ng / mL fibroblast growth factor 10 (Sino Biological) + 5 ng / mL fibroblast growth factor 2 (R&D) + 1 μmol / L prostaglandin E2 (Tocris) + 0.3 μmol / L CHIR99021 (purchased from MCE) + 1 μmol / L Forskolin (purchased from MCE) + 10 ng / mL R-spondin (purchased from R&D) + 10 ng / mL Noggin (purchased from R&D). Hereinafter referred to as ED medium.

[0136] (2) Oral cancer organoid culture

[0137] Oral cancer primary cells were obtained from intraoperative tissue sample OZ-022 according to the method of Example 1(2), and organoids were cultured in OC-3 medium and ED medium according to the method of Example 3.

[0138] On day 10 of culture, the cultured oral cancer organoids were observed using a microscope (Invitrogen EVOS M500). Figure 4A and 4B These are photographs of organoids cultured in OC-3 and ED media, taken under a 10x objective lens. Figure 4C This is a bar chart comparing the relative sizes of organoids obtained from two different culture media.

[0139] according to Figures 4A-4C The results showed that, compared with ED medium, OC-3 medium significantly promoted the expansion and culture of oral cancer organoids.

[0140] Example 5: Oral cancer organoids amplified using the culture medium of the present invention were used for drug screening.

[0141] (1) Oral cancer organoid culture

[0142] Oral cancer primary cells were isolated from the obtained oral cancer intraoperative sample (OZ-016) according to the method of Example 1 (2), and organoids were cultured using OC-3 medium. Drug screening was performed when the diameter of the oral cancer organoids exceeded 50 μm.

[0143] (2) Screening drug formulation

[0144] Prepare three drugs (daunorubicin, arubicin, and doxorubicin; all purchased from MCE) at two concentration gradients according to the table below and store them for later use.

[0145] Table 3. Preparation of Daunorubicin, Ararubicin, and Doxorubicin Drug Additives

[0146] Aroubi Xing Final concentration (μM) 1 0.1 Dorothy Star Final concentration (μM) 0.1 0.01

[0147] (3) Adding medicine

[0148] Take out the prepared drug and place it at room temperature. Dilute the drug 1000 times with OC-3 medium for later use. Take out the organoids obtained by culturing according to step (1) from the incubator, remove the culture medium from the culture wells, and slowly pour the culture medium containing the drug into the culture wells along the well walls. After the drug addition is completed, sterilize the surface of the 96-well plate and transfer it to the incubator for further culture. Measure the organoid viability after 72 hours.

[0149] (4) Organoid viability test

[0150] Remove CellTiter-Glo luminescent reagent (purchased from Promega) from the 4℃ freezer. Take 10 ml of the reagent into the sample container. Take the 96-well plate to be tested from the incubator and add 20 μL of CellTiter-Glo luminescent reagent to each well. Let stand for 10 minutes and then mix well. Use a multi-functional microplate reader (Perkin Elmer Envision) to detect the results.

[0151] (5) Data processing

[0152] According to the formula: Drug inhibition rate (%) = 100% - (Chemiluminescence value of culture well on the third day) 药物处理组 Chemiluminescence values ​​of culture wells on day zero 药物处理组 ) / (Chemiluminescence values ​​of culture wells on day 3) DMSO Chemiluminescence values ​​of culture wells on day zero DMSO The inhibition rate of different drugs was calculated by multiplying the result by 100%, and the results are shown below. Figure 5A and 5B . Figure 5A Images of organoid growth without drug treatment and images of organoid growth 3 days after drug treatment, taken under a 4x objective microscope (Invitrogen EVOS M500). Figure 5BBar chart showing the inhibition rate of different concentrations of the test drug on the growth of oral cancer organoids.

[0153] Depend on Figure 5A It can be confirmed that the organoids cultured using the oral cancer organoid culture medium of the present invention have good growth status, and the growth of organoids is significantly inhibited after treatment with daunorubicin and arubicin. Figure 5B This is a bar chart showing the inhibition rates of three test drugs at different concentrations in inhibiting the growth of oral cancer organoids. Figure 5B The data error values ​​for the drug treatment groups are very small, indicating that the data from replicate wells for the same drug are generally consistent when using this system for drug screening. Among the three antitumor drugs, arubicin showed strong inhibitory effects on organoid growth at both concentrations, while the inhibitory effects of different concentrations of daunorubicin varied significantly. Doxorubicin showed weaker inhibitory effects on organoid growth, suggesting that organoids from the same patient have different sensitivities to different drugs. Based on these results, the effectiveness and optimal dosage of this drug in clinical use for oral cancer patients can be determined.

Claims

1. A culture medium for oral cancer organoids, characterized in that... Includes MST1 / 2 kinase inhibitors, at least one cell culture additive selected from N2 and B27, fibroblast growth factor, CHIR99021, epidermal growth factor, insulin, ITS cell culture additive, SB202190, Y27632, dexamethasone, GlutaMAX, and non-essential amino acids. The culture medium does not contain Wnt agonists, R-spondin family proteins, Noggin protein, BMP inhibitors, or fibroblast growth factor 10. The MST1 / 2 kinase inhibitor is selected from compound 1 or its pharmaceutically acceptable salt or solvate. Compound 1 in, The concentration of the MST1 / 2 kinase inhibitor is 2.5–10 μM; The volume ratio of the B27 or N2 cell culture additive to the culture medium is 1:25 to 1:

100. The concentration of the fibroblast growth factor is 3–30 ng / mL; The concentration of CHIR99021 is 1.25–5 μM; The concentration of the epidermal growth factor is 2–18 μM; The concentration of the insulin is 1–10 μg / mL; The volume ratio of the ITS cell culture additive to the culture medium is 1:30 to 1:300; The concentration of SB202190 is 100–400 nM; The concentration of Y27632 is 3–30 μM; The concentration of dexamethasone was 0.02–0.5 μM; The volume ratio of GlutaMAX to the culture medium is 1:30 to 1:300; The non-essential amino acid is selected from one or more of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine, and its concentration is 50–200 μM.

2. The culture medium as described in claim 1, characterized in that... Also includes: Initial culture media selected from DMEM / F12, DMEM, F12, or RPMI-1640; and An antibiotic selected from one or more of streptomycin / penicillin, amphotericin B, and primocin.

3. A method for culturing oral cancer organoids, characterized in that... Includes the following steps: (1) Separate samples from oral cancer solid tumor tissue to obtain primary oral cancer cells; (2) Prepare the culture medium for oral cancer organoids according to claim 1 or 2, and perform organoid culture on the primary oral cancer cells obtained in step (1).

4. A method for evaluating or screening drugs for treating oral cancer, characterized in that, Includes the following steps: (1) Oral cancer organoids are cultured using the oral cancer organoid culture method of claim 3; (2) Select the drug to be tested and dilute it according to the required concentration gradient; (3) Add the diluted drug to the organoids cultured in (1); (4) Perform organoid size or organoid viability testing.

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