NUT cancer organoid as well as culture method, culture medium and application thereof

Abstract

The invention discloses an NUT cancer organ as well as a culture method, a culture medium and application thereof, and belongs to the technical field of biological medicines. The culture medium comprises 10 to 100 [mu] M / L of GSK269962A, 1 to 10 nM / L of trametinib and 1 to 10 [mu] M / L of BIP-135. The invention also provides a culture method of the NUT cancer organoid. The culture method comprises the following steps: obtaining a pleuroperitoneal fluid sample of an NUT cancer patient, carrying out anticoagulation treatment, and centrifuging to obtain cell precipitate; re-suspending the cell precipitate with PBS, and enriching the cells through Percoll density gradient centrifugation; and mixing the enriched cells with matrigel, inoculating the mixture to a culture plate, adding a culture medium, and culturing for 5-7 days to obtain the NUT cancer organoid. The cultured NUT cancer organoid can be used for screening drugs for treating NUT cancer and detecting the sensitivity of anti-cancer drugs to NUT cancer, is especially suitable for sensitivity detection of NHWD-870 drugs, and provides a new research tool for individualized treatment of NUT cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a NUT carcinoma organoid, a culture method, a culture medium and an application thereof. BACKGROUND

[0002] NUT carcinoma (NC) is a rare and highly aggressive malignancy caused by chromosomal translocation of NUTM1 gene with other genes (such as BRD4, BRD3 or NSD3). The prognosis of this disease is extremely poor, with a median survival of only 6-7 months, and it is not responsive to traditional chemotherapy and radiotherapy. Currently, the diagnosis and treatment of NUT carcinoma still face great challenges, and new research models and treatment strategies need to be developed urgently.

[0003] Tumor organoids, as a new three-dimensional culture technology, can maintain the heterogeneity of tumors and the genomic characteristics of primary tumors in vitro, and have been widely used in the study of various tumors. For example, CN112592896B discloses a culture medium and a culture method for lung adenocarcinoma organoids, which comprises a basic culture medium and specific additives, and can improve the culture success rate and the number of passages of the organoids. CN119286789A provides a special culture medium for breast cancer organoids and a culture method thereof, which is composed of DMEM / F12 basic culture medium and various culture additives, and can reduce the cost of culture while improving the stability of tumor cell genome during culture.

[0004] Currently, there is no specific organoid culture system for NUT carcinoma, and the existing culture methods cannot meet the culture needs of NUT carcinoma, which is a rare and highly aggressive tumor. Secondly, there are differences in drug response among NUTM1 fusion gene type NUT carcinoma patients, and there is a lack of effective in vitro drug sensitivity detection method to screen potential responsive patient population. Thirdly, there is no report on establishing tumor organoids based on NUT carcinoma pleural effusion in the prior art, and pleural effusion samples are more convenient to obtain and less invasive than tissue biopsy, which is particularly suitable for the study of rare tumors. In addition, there is a lack of specific signal pathway regulating factors for NUT carcinoma in the existing culture system, which cannot effectively maintain the growth and characteristics of NUT carcinoma organoids.

[0005] Therefore, it is urgent to develop an organoid culture medium and culture method specifically for NUT carcinoma to establish a stable NUT carcinoma organoid model and provide an effective tool for drug screening and individualized treatment of NUT carcinoma. SUMMARY

[0006] In order to solve the technical problems existing in the treatment of NUT cancer, and realize the purpose of individualized treatment for NUT cancer patients, the application provides a NUT cancer organoid and its culture method, culture medium and application. The technical problems to be solved by the application are that NUT cancer is a rare and highly invasive cancer, which is not sensitive to traditional treatment and has a poor prognosis; NUT cancer patients with different NUTM1 fusion gene types have different responses to drugs, and it is necessary to screen out a patient population that responds; there is no report on establishing a tumor organoid based on NUT cancer pleural effusion, and there is a lack of effective in vitro drug sensitivity detection method.

[0007] The technical scheme adopted by the application to solve its technical problems is as follows: Firstly, a culture medium for culturing NUT cancer organoids is provided, characterized in that it comprises 10-100 μM / L GSK269962A, 1~10 nM / L Trametinib and 1~10 μM / L BIP-135.

[0008] GSK269962A as a ROCK inhibitor can inhibit the activity of Rho-related protein kinase, reduce apoptosis and promote the survival and proliferation of NUT cancer cells. Its concentration can be selected from 10-100 μM / L, such as 10 μM / L, 25 μM / L, 50 μM / L, 75 μM / L or 100 μM / L, etc., which will not be repeated here. Preferably, the concentration of GSK269962A is 50 μM / L.

[0009] Trametinib as a MEK1 / 2 inhibitor (mitogen-activated protein kinase inhibitor) can inhibit the MAPK signaling pathway and regulate the proliferation and differentiation of NUT cancer cells. Its concentration can be selected from 1~10 nM / L, such as 1 nM / L, 2.5 nM / L, 5 nM / L, 7.5 nM / L or 10 nM / L, etc., which will not be repeated here. Preferably, the concentration of Trametinib is 5 nM / L.

[0010] BIP-135 is a highly selective ATP-competitive glycogen synthase kinase-3 (GSK-3) inhibitor, and its concentration can be selected from 1~10 μM / L, such as 1 μM / L, 2.5 μM / L, 5 μM / L, 7.5 μM / L or 10 μM / L, etc., which will not be repeated here. Preferably, the concentration of BIP-135 is 5 μM / L.

[0011] The three inhibitors GSK269962A, Trametinib and BIP-135 have a synergistic effect after combination, and can significantly improve the culture efficiency and stability of the NUT cancer organoids.

[0012] Preferably, the culture medium further comprises a basic culture medium Advanced DMEM / F-12, a B27 supplement, antibiotics, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and the antibiotics are penicillin and streptomycin.

[0013] Secondly, the application provides a culture method of NUT cancer organoids, which uses the culture medium of the NUT cancer organoids to culture NUT cancer ascites cells to obtain the NUT cancer organoids.

[0014] Preferably, the culture method comprises the following steps: (1) obtaining a NUT cancer patient ascites sample, centrifuging the sample after anticoagulation treatment to obtain a cell precipitate; (2) resuspending the cell precipitate with phosphate buffered saline (PBS), superimposing the cell precipitate on Percoll density gradient liquid, centrifuging, and collecting the interface layer cells; (3) resuspending the enriched cells in the NUT cancer organoid culture medium at a concentration of 1×10 5 cells / mL, mixing the cells with Matrigel at a volume ratio of 1:1, inoculating the mixture into a culture plate, incubating, adding the NUT cancer organoid culture medium to cover the gel after the Matrigel solidifies, and continuing to culture for 5-7 days to obtain the NUT cancer organoids.

[0015] Preferably, in step (1), the anticoagulation treatment uses 4% sodium citrate, and the addition amount is 1 / 10 of the volume of the ascites; and the centrifugation condition is 600g centrifugation at room temperature for 5 minutes.

[0016] Preferably, in step (2), the Percoll density gradient liquid is a mixture of Percoll and phosphate buffered saline (PBS), the density range is 1.055-1.060g / ml, the centrifugation speed is 400-600g, and the centrifugation time is 15-20 minutes.

[0017] Preferably, in step (3), the incubation is to place the inverted culture plate in a 37℃ incubator for 30 minutes.

[0018] The application further provides a NUT cancer organoid, which is obtained by the culture method.

[0019] The application further provides an application of the NUT cancer organoid in detecting the sensitivity of NUT cancer to anticancer drugs. Preferably, the drug is NHWD-870.

[0020] Finally, the application also provides the use of the NUT cancer organoids in screening drugs for treating NUT cancer.

[0021] The application has the following advantages: 1. The application can enrich NUT cancer cells in pleural effusion or ascites by density gradient centrifugation, the density of the density gradient separation solution is 1.055-1.060 g / ml, the centrifugal speed is 400-600 g, and the centrifugal time is 15-20 minutes, which can effectively enrich NUT cancer cells in pleural effusion or ascites, and provide a high-quality cell source for subsequent organoid culture.

[0022] 2. The NUT cancer organoid culture medium of the application contains a combination of GSK269962A (a ROCK inhibitor), Trametinib (a MEK1 / 2 inhibitor) and BIP-135 (a GSK-3 inhibitor), and the combination of the three inhibitors has a synergistic effect, and the number of NUT cancer organoids in the culture is significantly higher than that of single use, which provides better conditions for the culture of NUT cancer organoids.

[0023] 3. The application successfully obtains NUT cancer organoids, which express positive NUTM1 fusion protein and contain different types of NUTM1 gene fusions, proving the effectiveness and reliability of the culture method of the application.

[0024] 4. The application adds NUT cancer organoid culture medium containing solvent or NHWD-870 to treat NUT cancer organoids, wherein the final concentration of NHWD-870 is 1 / 10 of its peak plasma concentration, i.e. 3.2 ng / mL, and the antitumor effect of NHWD-870 on NUT cancer organoids is divided into four levels according to cell survival rate: sensitive (≤10%), partially sensitive (11%-50%), slightly sensitive (51%-75%) and resistant (75%), and the NUT cancer organoid drug sensitivity test can effectively predict the clinical response of patients to NHWD-870, and the in vitro drug sensitivity results are consistent with the clinical research results, which provides an important basis for individualized treatment of NUT cancer patients.

[0025] 5. The culture method of the application can quickly and effectively obtain NUT cancer tumor organoids derived from pleural effusion or ascites, which provides a better tool for studying individualized treatment of NUT cancer, helps to screen NUT cancer patient groups sensitive to anticancer drugs (such as NHWD-870), improves treatment effect, and improves patient prognosis. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The culture effect of different inhibitors alone or in combination on NUT cancer organoids. Among them, Figure 1A is the photo of NUT cancer organoids NUT05 after the effect of different inhibitors, Figure 1 B is the quantitative result of NUT cancer organoids NUT05 culture after the effect of different inhibitors. Statistical processing uses One-way ANOVA, *P<0.001, comparison between additive group and solvent control; **P<0.001, comparison between additive combination and single use.

[0027] Figure 2 It is the morphological picture of NUT cancer organoids of 4 samples in Example 4 and the picture of NUT fusion protein expression detected by immunoblotting method; wherein, Figure 2 A is the photo of different NUT cancer organoids cultured, Figure 2 B is the picture of NUT fusion protein expression detected by immunoblotting method; Figure 3 It is a flowchart of NHWD-870 drug sensitivity detection of NUT cancer organoids; Figure 4 It is the cell survival rate of NUT cancer organoids after the effect of NHWD-870 detected by CCK-8; Figure 5 It is the tumor image of NUT cancer patient sensitive to NHWD-870 after oral administration of NHWD-870 for treatment. DETAILED DESCRIPTION

[0028] The present application will be further described in detail below in combination with the drawings and examples, but in no way limits the present application, any transformation or improvement based on the teaching of the present application falls within the protection scope of the present application.

[0029] The process, condition, reagent, experimental method, etc. for implementing the present application, except for the following specially mentioned contents, are the general knowledge and common sense in the art, and the present application has no special limitation. The experimental method without specific condition in each example is usually according to the conventional condition or according to the condition suggested by the manufacturer.

[0030] The following are the sources of some reagents and materials in the examples: GSK269962A (MedChemExpress, Catalog No. HY-15556 purity: 99.83%); Trametinib (MedChemExpress, Catalog No. HY-10999 purity: 99.93%); BIP-135 (MedChemExpress, Catalog No. HY-111055 purity: 99.86%); NHWD-870 (MedChemExpress, Catalog No. HY-134463 purity: 99.36%); The pleural effusion samples of NUT cancer patients come from the NUT cancer specialist of Chongqing University Affiliated Hospital, and all patients have given their informed consent.

[0031] Embodiment 1 The culture medium for culturing NUT cancer organoids provided in this embodiment comprises 50 μM / L GSK269962A, 5 nM / L Trametinib and 5 μM / L BIP-135, a basic culture medium Advanced DMEM / F-12 (98% of the total volume of the culture medium by volume), B27 supplement (2% of the total volume of the culture medium by volume), the final concentration of EGF is 50 ng / mL, the final concentration of bFGF is 20 ng / mL, the final concentration of penicillin is 100 U / mL, and the final concentration of streptomycin is 100 μg / m.

[0032] The preparation method of the culture medium comprises the following steps: uniformly mixing the basic culture medium Advanced DMEM / F-12, B27 supplement, penicillin and streptomycin, EGF and bFGF, then adding GSK269962A, Trametinib and BIP-135, adjusting the pH value to 7.2-7.4, and filtering through a 0.22 μm filter membrane to remove bacteria.

[0033] Embodiment 2 The difference between this embodiment and Embodiment 1 is that the concentrations of GSK269962A, Trametinib and BIP-135 are 100 μM / L, 10 nM / L and 10 μM / L respectively, and the rest is the same as Embodiment 1.

[0034] Embodiment 3 The difference between this embodiment and Embodiment 1 is that the concentrations of GSK269962A, Trametinib and BIP-135 are 10 μM / L, 1 nM / L and 1 μM / L respectively, and the rest is the same as Embodiment 1.

[0035] Embodiment 4 The culture method of NUT cancer organoids provided in this embodiment uses the culture medium of NUT cancer organoids described in Embodiment 1 to culture NUT cancer pleural effusion cells, so as to obtain NUT cancer organoids.

[0036] The culture method comprises the following steps: Step 1: Obtain the pleural effusion samples of NUT cancer patients (NUT05, NUT07, NUT08 and NUT09), centrifuge the cell precipitate after anticoagulation treatment.

[0037] Specifically, the anticoagulation treatment uses 4% sodium citrate, and the addition amount is 1 / 10 of the volume of the pleural effusion; the centrifugation condition is 600g centrifugation at room temperature for 5 minutes.

[0038] Step two: Resuspend the cell pellet with PBS, overlay on Percoll density gradient, centrifuge, collect the interface layer cells.

[0039] Specifically, the Percoll density gradient is a mixture of Percoll and PBS with a density of 1.056 g / mL, and the centrifugation condition is 500 g for 15 minutes.

[0040] Step three: Resuspend the enriched cells in the NUT cancer organoid culture medium described in Example 1 at a concentration of 1 x 10 5 Incubate, after the matrigel solidifies, add the NUT cancer organoid culture medium described in Example 1 to cover the gel, continue to culture for 5-7 days to obtain NUT cancer organoids.

[0041] Specifically, incubate the inverted culture plate at 37°C for 30 minutes.

[0042] Example 5, which differs from Example 4 in that the Percoll density gradient has a density of 1.055 g / mL, and the centrifugation condition is 600 g for 20 minutes, and the rest is the same as Example 4.

[0043] Example 6, which differs from Example 4 in that the Percoll density gradient has a density of 1.060 g / mL, and the centrifugation condition is 600 g for 15 minutes, and the rest is the same as Example 4.

[0044] Comparative Example 1, which differs from Example 4 in that the inhibitor in the culture medium only contains 5.055 nM / L GSK269962A, and the NUT cancer patient ascites sample only contains NUT05, and the rest is the same as Example 4.

[0045] Comparative Example 2, which differs from Example 4 in that the inhibitor in the culture medium only contains 5.055 nM / L Trametinib, and the NUT cancer patient ascites sample only contains NUT05, and the rest is the same as Example 4.

[0046] Comparative Example 3, which differs from Example 4 in that the inhibitor in the culture medium only contains 5.055 nM / L BIP-135, and the NUT cancer patient ascites sample only contains NUT05, and the rest is the same as Example 4.

[0047] Detect the state and number of NUT cancer organoids in Example 4 (NUT cancer patient ascites sample is NUT05) and Comparative Examples 1-3. Count the number of organoids with a diameter greater than 50 μm under a 10x objective, count 5 fields of view in each group, calculate the mean and standard deviation. The number of organoids in the solvent control is 100%.

[0048] Test results are shown Figure 1 The results showed that the combined application of GSK269962A, Trametinib and BIP-135 exhibited the best support effect, with the number of NUT cancer organoids in the culture being significantly higher than that of the single application.

[0049] Identification of the NUT cancer organoids prepared in Example 4: The expression of the NUTM1 fusion protein was analyzed using Western blotting and immunofluorescence staining. Results are shown below. Figure 2 The results showed that NUT cancer organoids were successfully obtained, and their NUTM1 fusion protein expression was positive.

[0050] RNA was extracted from NUT cancer organoids using the RNAeasy™ Animal RNA Extraction Kit (Beyotime, R0026), and RNA sequencing analysis was performed. Table 1 shows the basic information and NUTM1 gene fusion types of NUT cancer patients with pleural and peritoneal effusions. The results showed that NUT cancer organoids contained different types of NUTM1 gene fusions.

[0051] Table 1. Basic information and NUTM1 gene fusion types of patients with NUT cancer organoids.

[0052] Example 7: Application of NUT cancer organoids in evaluating the sensitivity of anti-NUT cancer drugs NHWD-870 is the inventor's investigational drug that targets BRD4 to exert anti-tumor effects. However, NUT cancer patients with different NUTM1 fusion gene types respond differently to this drug. In this example, NUT cancer organoids prepared in Example 4 are used to evaluate the sensitivity of NHWD-870 to NUT cancer. The schematic diagram of the detection process is shown below. Figure 3 .

[0053] 1. Drug processing and culture of NUT cancer organoids Take 100 μL of cell suspension (concentration of 1×10⁻⁶) 5 (Prepared in Example 4), add 100 μL of pre-cooled matrix gel and mix well. Add 20 μL to each well of a 24-well plate, invert the plate and incubate at 37°C for 30 minutes. After the matrix gel solidifies, add NUT cancer culture medium containing solvent or NHWD-870 (NHWD-870 concentration 3.2 ng / mL) to each well, and set up a cell-free control group. Continue culturing for 5–7 days.

[0054] 2. Evaluation of the antitumor effect of drugs Add 50 μL of CCK-8 stock solution to each well of the 24-well plate and incubate at 37°C for 4 hours. Read the OD values ​​using a microplate reader. 450, calculate cell survival rate = (NHWD-870 group OD 450 - blank OD 450 ) / (solvent group OD 450 - blank OD 450 ) x 100%. The antitumor effect of NHWD-870 is divided into four levels: sensitive: cell survival rate ≤ 10%; partially sensitive: cell survival rate between 11% and 50%; slightly sensitive: cell survival rate between 51% and 75%; resistant: cell survival rate greater than 75%.

[0055] The results are shown in Figure 4 , NUT cancer organoid drug sensitivity test found that NHWD-870 in vitro antitumor effect evaluation of NUT05 and NUT09 is sensitive, after treatment with NHWD-870, its cell survival was 8.2% and 6.4% respectively; NHWD-870 in vitro antitumor effect evaluation of NUT07 and NUT08 is slightly sensitive and resistant, after treatment with NHWD-870, its cell survival was 73.2% and 93.6% respectively.

[0056] 3. Consistency of drug sensitivity test results and clinical research As shown in Figure 4 , after two months of NHWD-870 (1.5 mg / day) administration, CT examination showed that the tumor of NUT05 and NUT09 patients was significantly reduced compared with the baseline, and the efficacy was evaluated as partial remission according to RECIST standard.

[0057] The above results show that the NUT cancer organoid drug sensitivity test method can quickly evaluate the therapeutic effect of anticancer drugs on NUT cancer, provide a scientific basis for individualized drug use in clinic, and help to improve the treatment effect and survival rate of NUT cancer patients.

[0058] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. However, if there is a conflict between the definitions of terms in the specification and the definitions of terms contained in the included definitions, the definitions contained in the specification shall prevail. Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the same; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent substitutions for some of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A culture medium for culturing NUT carcinoma organoids, characterized by, comprising 10-100 μM / L GSK269962A, 1-10 nM / L trametinib and 1-10 μM / L BIP-135.

2. The medium according to claim 1, characterized in that, The medium further comprises basal medium Advanced DMEM / F-12, B27 supplement, antibiotics, epidermal growth factor, basic fibroblast growth factor, and the antibiotics are penicillin and streptomycin.

3. A method for culturing NUT carcinoma organoids, characterized by, The NUT carcinoma pleural effusion cancer cells are cultured by using the medium of the NUT carcinoma organoid according to any one of claims 1-2, so as to obtain the NUT carcinoma organoid.

4. The culturing method according to claim 3, wherein, comprising the following steps: (1) obtaining a NUT carcinoma patient pleural effusion sample, centrifuging the sample after anticoagulation to obtain a cell precipitate; (2) resuspending the cell precipitate with a phosphate buffer, superimposing the cell precipitate on a Percoll density gradient liquid, centrifuging, and collecting the interface layer cells; (3) Resuspend the enriched cells at a concentration of 1 x 10 5 cells / mL in the NUT cancer organoid culture medium of any one of claims 1 or 2, mix with Matrigel at a 1 : 1 volume ratio, seed the mixture into a culture plate, incubate, and after the Matrigel has solidified, overlay the Matrigel with the NUT cancer organoid culture medium of any one of claims 1 or 2, and continue culturing for 5-7 days to obtain the NUT cancer organoid.

5. The method of claim 4, wherein: In step (1), the anticoagulation treatment uses 4% sodium citrate, and the addition amount is 1 / 10 of the volume of the pleural effusion; the centrifugation condition is 600g centrifugation at room temperature for 5 minutes.

6. The method of claim 4, wherein: In step (2), the Percoll density gradient liquid is a mixture of Percoll and phosphate buffer, the density range is 1.055-1.060 g / ml, the centrifugation speed is 400-600g, and the centrifugation time is 15-20 minutes.

7. The method of claim 4, wherein: In step (3), the incubation is to place the inverted culture plate at 37°C for 30 minutes.

8. A NUT cancer organoid, characterized in that, The NUT carcinoma organoid is obtained by the culture method according to any one of claims 3-7.

9. The NUT carcinoma organoid of claim 8 is used for screening drugs for treating NUT carcinoma.

10. The NUT carcinoma organoid of claim 8 is used for detecting the sensitivity of anti-cancer drugs to NUT carcinoma. Preferably, the drug is NHWD-870.

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