Improved construction method of ovarian cancer organoid and application of improved construction method in natural products

Abstract

The invention discloses an improved construction method of ovarian cancer organoid and application of the ovarian cancer organoid in natural products, and relates to the technical field of medical biology. The method comprises the following steps: cutting sterile cleaned ovarian cancer tissues into milk, digesting, sieving, collecting filtrate, repeating for 2-5 times, then adding into an ovarian cancer organoid culture medium for culturing, selecting stably growing ovarian cancer organoid to screen a natural product, adding the natural product for culturing for 4 days, and detecting the cell activity to obtain the ovarian cancer targeted natural product. By optimizing the culture of the ovarian cancer organoid, the success rate and passage capacity of the culture of the organoid are improved, the modeling period is shortened, and the tumor state can be simulated to screen natural product drugs in vitro.

Description

Technical Field

[0001] The present invention relates to the field of medical biotechnology, and in particular to an improved construction method of ovarian cancer organoids and application thereof in natural products. Background Art

[0002] Ovarian cancer is one of the most common diseases in women. It ranks fifth in the number of deaths among female cancers, higher than other types of cancers of the female reproductive system, and has a five-year survival rate of less than 50%. Currently, the first-line treatment for ovarian cancer is still tumor cell reduction surgery and postoperative adjuvant chemotherapy with a combination of platinum and paclitaxel. Although chemotherapy can improve the prognosis of ovarian cancer to a certain extent, the five-year survival rate of patients with advanced ovarian cancer is still less than 30%. After the initial treatment, more than 70% of patients with advanced ovarian cancer experience recurrence and platinum resistance. High recurrence rate and chemotherapy resistance are the main causes of death in ovarian cancer patients. In recent years, although new drugs for tumors have emerged in an endless stream, only 20% have finally entered the clinic, and they are extremely expensive. Preclinical models that accurately simulate the biological characteristics of ovarian cancer in vivo are essential.

[0003] Most current preclinical in vitro studies of tumors rely on traditional two-dimensional planar culture systems. However, due to the significant differences between such culture conditions and the in vivo environment, the cultured cells or tissues lose their original tumor morphology, structure and function, and also lose the heterogeneity of the tumor. Organoids are tissue analogs with specific spatial organizations created by in vitro three-dimensional stem cell culture. They can simulate or reproduce a series of physiological and pathological processes in vivo in vitro and have relatively stable phenotypes and genetic characteristics. In precision medicine applications, tumor organoids derived from tumor patients have been shown to be valuable diagnostic tools. In the ovarian tumor organoid culture system, after adding various growth factors and matrix components of the in vivo microenvironment, a culture system close to the in vivo tumor microenvironment can be formed. Cells derived from individual tissues of ovarian cancer patients form patient-derived ovarian cancer organoids, which have similar histological and genomic characteristics compared with the patient's autologous tumor.

[0004] Natural products, due to their structural diversity and biological activity, have always occupied a special position in the human fight against cancer as an important source of anticancer drug research and development. From paclitaxel, vincristine to artemisinin derivatives, natural active ingredients continue to refresh the cognitive boundaries of tumor treatment. However, traditional in vitro cell experiments often cannot accurately evaluate their effects in complex tumor microenvironments, and animal models also have challenges in species differences and ethical issues. Therefore, evaluating the efficacy of natural products in the ovarian cancer organoid (OCO) model can more accurately screen candidate compounds with clinical transformation potential and improve the success rate of drug development. This strategy not only helps to deeply analyze the mechanism of action of natural products, but also promotes the development of personalized treatment and provides more effective treatment options for ovarian cancer patients. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide an improved construction method of ovarian cancer organoids and their application in natural products. By optimizing the culture of ovarian cancer organoids, the success rate and propagation ability of organoid culture are improved, the modeling cycle is shortened, and the tumor state can be simulated to perform natural product drug screening in vitro.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: providing the application of ovarian cancer organoids in the preparation of anti-tumor natural products.

[0007] Furthermore, the natural product is pristmerin.

[0008] At the same time, the present invention also provides an improved method for constructing ovarian cancer organoids, comprising the following steps:

[0009] The sterile cleaned ovarian cancer tissue is chopped into a milky state, and the filtrate is collected by sieving after digestion, and repeated 2-5 times. The ovarian cancer organoid culture medium is then added to culture, and the stable growing ovarian cancer organoids are selected to screen natural products, and the natural products are added to culture, and the cell viability is detected to obtain ovarian cancer-targeted natural products.

[0010] Furthermore, the cells were digested in 1 mg / mL collagenase IV for 10 min and the filtrate was collected through a 100 μm cell sieve.

[0011] Further, the ovarian cancer organoid culture medium includes the following components: recombinant human R-Spondin-1 protein 500-1000 ng / ml, recombinant human Noggin protein 1-100 ng / ml, recombinant human FGF10 protein 1-20 ng / ml, recombinant human EGF protein 1-10 ng / ml, recombinant human Heregulin β-1 protein 1-50 ng / ml, nicotinamide 1-15 μM, forskolin 10-20 μM, hydrocortisone 1-500 ng / ml, estradiol 50-100 nM, Y27632 1-5 μM, N-acetylcysteine ​​1-1.25 mM, B27 1×, A83-01 0.1-0.5 μM, GlutaMAX supplement 1×, HEPES buffer 1×, Primocin 0.2%-0.4%.

[0012] Furthermore, after adding the natural product and mixing, 20 μL was dropped into each well of a preheated 48-well plate and placed in a 37°C incubator for 20 min to solidify, and 250 μL of ovarian cancer organoid culture medium was added to each well, and the plates were cultured in a 37°C, 5% carbon dioxide incubator.

[0013] Furthermore, Calcein / PI detection reagent was used to detect cell viability.

[0014] Furthermore, the ovarian cancer targeting natural product is prismaticin.

[0015] The present invention has the following beneficial effects:

[0016] 1. The present invention provides an optimized culture method for ovarian cancer organoids with a higher success rate, and uses ovarian cancer organoids for natural product screening, utilizing the tumor microenvironment to simulate the in vivo tumor state for high-throughput drug screening.

[0017] 2. This culture method improves the success rate and propagation capacity of organoid culture, shortens the modeling cycle, and can simulate tumor states to screen natural product drugs in vitro, making the evaluation of the anti-tumor activity of natural products more clinically relevant and helpful for screening candidate compounds for different molecular subtypes. Secondly, organoids can maintain key cell-to-cell interactions in the tumor microenvironment, including the coexistence of tumor cells and stromal cells, thereby more accurately evaluating the effects of natural products on the tumor microenvironment. In addition, compared with traditional two-dimensional cell culture, the simulation of organoid drug response is closer to the in vivo situation, and the anti-cancer activity and drug resistance of natural products can be more accurately predicted. At the same time, compared with animal models, organoid culture has higher repeatability and scalability, reduces the interference caused by species differences, and meets ethical requirements. Therefore, ovarian cancer organoids not only improve the screening efficiency in the efficacy evaluation of natural products, but also provide a more reliable research platform for personalized treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of ovarian cancer OCO1-4 organoids at different growth days;

[0019] Figure 2 Schematic diagram of ovarian cancer OCO1-3 P0 and P5 organoids;

[0020] Figure 3 This is a Calcein / PI staining image of ovarian cancer organoids for the natural product pristmerin;

[0021] Figure 4 This figure shows the effect of the natural product pristmerin on the cell activity of ovarian cancer organoids. DETAILED DESCRIPTION

[0022] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0023] Example 1

[0024] An improved method for constructing ovarian cancer organoids, comprising the following steps:

[0025] (1) Ovarian cancer tissue processing

[0026] a. Freshly obtained ovarian cancer tissues were stored in tissue preservation solution and transported to the laboratory on ice;

[0027] b. Place in a sterile culture dish, rinse twice with PBS and once with AdDF+++, cut with sterile scissors, and select one piece for organoid construction;

[0028] c. Ovarian cancer tissue used for organoid construction was minced into milky pieces, 1 mm 3 left and right size;

[0029] d. Add 10 mL of AdDF+++ to the washed tissue and place it in a centrifuge tube for 5-10 minutes. Let the larger pieces of tissue settle by gravity and collect the supernatant into another centrifuge tube;

[0030] e. The precipitated larger tissues were digested in 1 mg / mL collagenase IV. After 10 min of digestion, the tissues were passed through a 100 μm cell sieve and the filtrate was combined with the above supernatant.

[0031] f. Continue digesting the large tissue fragments on the filter for 10 minutes, repeat this step twice until the tissue is completely digested, and combine the filtrates. This step is the preferred method for obtaining primary ovarian cancer cells;

[0032] g. Centrifuge the filtrate at 1000 rpm for 5 min. If there are a lot of red blood cells, add 2 mL of red blood cell lysis buffer to lyse for 5 min, add 10 mL of AdDF+++ to wash, centrifuge at 1000 rpm for 5 min, and remove the supernatant.

[0033] (2) Ovarian cancer organoid culture

[0034] According to the amount of cell precipitation, a certain volume of ovarian cancer organoid culture medium and Matrigel mixture is added. The preferred ratio of ovarian cancer organoid culture medium and Matrigel in the present invention is 7:3. Gently mix evenly, drop 20 μL into each well of a preheated 48-well plate, place in a 37°C incubator for 20 minutes to solidify, and then add 250 μL of ovarian cancer organoid culture medium to each well, and culture in a 37°C, 5% carbon dioxide incubator. Primary OCO1, OCO2, OCO3, OCO4 of different growth days and OCO1, OCO2, OCO3 of P0 and P5, respectively, are as follows Figure 1-2 shown.

[0035] The components and concentrations of ovarian cancer culture medium are as follows:

[0036] Recombinant Human R-Spondin 1Protein: 500ng / ml, Wnt / βcatenin signaling pathway regulator;

[0037] Recombinant Human Noggin Protein: 100 ng / ml, BMPs regulator;

[0038] B27 1×, cell culture supplement;

[0039] Nicotinamide: 10 μM, the active components of NAD and NADP;

[0040] Recombinant Human FGF-10 Protein (recombinant human FGF10 protein): 10ng / ml, Recombinant Human EGF Protein (recombinant human EGF protein): 5ng / ml,

[0041] Forskolin: 10 μM, activates cAMP;

[0042] Hydrocortisone: 500 ng / ml

[0043] β-Estradiol: 100nM

[0044] Y27632: 5 μM, prevents stem cell apoptosis;

[0045] Human Neuregulin-1 (NRG1) (recombinant human Neuregulin β-1 protein): 37.5 ng / ml;

[0046] N-Acetylcysteine: 1.25mM, ROS inhibitor;

[0047] A83-01: 0.5 μM, ALK inhibitor;

[0048] Advanced DMEM / F12 (modified DMEM basal medium)

[0049] Primocin 0.2%;

[0050] GlutaMAX supplement: 1×;

[0051] HEPES (n-2-hydroxyethylpiperidin-n-2-ethanesulfonic acid) buffer: 1×;

[0052] (3) Natural product screening

[0053] Stably growing ovarian cancer organoids were selected and re-inoculated into 96-well black transparent glass plates at 10 μL per well. The culture plates were divided into a control group and an experimental group. 24 hours after inoculation, ovarian cancer organoid culture medium containing 0.1% DMSO was added to the control group, and natural products, such as pristmerin at concentrations of 0.25 μM and 0.5 μM, were added to the experimental group and cultured for 4 days. The supernatant culture medium was then discarded and the plates were rinsed once with PBS buffer (natural products can be detected after incubating ovarian cancer organoids for 4 days).

[0054] The staining experiment was performed according to the operating instructions of the Calcein / PI cell activity and cytotoxicity detection kit. 100 μL of Calcein / PI detection working solution was added to each well, and the cells were placed in a constant temperature incubator at 37°C and 5% carbon dioxide for incubation for 30 min. The cells were washed with PBS three times, each time for 3-5 min, and fluorescent images were acquired and analyzed using ImageXpress Micro 4 (Molecular Devices, American).

[0055] Calcein / PI staining of the above ovarian cancer organoids for the natural product pristmerin is shown below Figure 3 As shown in Figure 2, the natural product pristmerin affects the cell viability of ovarian cancer organoids. Figure 4shown.

[0056] Depend on Figure 3 It can be seen that the number of dead cells in the ovarian cancer organoids in the experimental group increased significantly; combined with Figure 4 The quantitative analysis results showed that pristmerin promoted the apoptosis of ovarian cancer organoids. In summary, the results showed that pristmerin had an inhibitory effect on ovarian cancer organoids in a dose-dependent manner.

[0057] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of ovarian cancer organoids in the preparation of anti-tumor natural products.

2. The improved method for constructing ovarian cancer organoids according to claim 1, characterized in that: The following steps are involved: The sterile cleaned ovarian cancer tissue is chopped into a milky state, and the filtrate is collected by sieving after digestion, and repeated 2-5 times. The ovarian cancer organoid culture medium is then added to culture, and the stable growing ovarian cancer organoids are selected to screen natural products, and the natural products are added to culture, and the cell viability is detected to obtain ovarian cancer-targeted natural products.

3. The improved method for constructing ovarian cancer organoids according to claim 2, characterized in that: After digestion in 1 mg / mL collagenase IV for 10 min, the filtrate was collected by passing through a 100 μm cell sieve.

4. The improved method for constructing ovarian cancer organoids according to claim 2, characterized in that: The ovarian cancer organoid culture medium includes the following components: recombinant human R-Spondin 1 protein 500-1000 ng / ml, recombinant human Noggin protein 1-100 ng / ml, recombinant human FGF10 protein 1-20 ng / ml, recombinant human EGF protein 1-10 ng / ml, recombinant human Heregulin β1 protein 1-50 ng / ml, nicotinamide 1-15 μM, forskolin 10-20 μM, hydrocortisone 1-500 ng / ml, estradiol 50-100 nM, Y27632 1-5 μM, N-acetylcysteine ​​1-1.25 mM, B27 1×, A83-01 0.1-0.5 μM, GlutaMAX supplement 1×, HEPES buffer 1×, and Primocin 0.2%-0.4%.

5. The improved method for constructing ovarian cancer organoids according to claim 2, characterized in that: After adding the natural product and mixing, 20 μL was dropped into each well of the preheated 48-well plate and placed in a 37°C incubator for 20 min to solidify. 250 μL of ovarian cancer organoid culture medium was added to each well and cultured in a 37°C, 5% carbon dioxide incubator.

6. The improved method for constructing ovarian cancer organoids according to claim 2, characterized in that: Calcein / PI detection reagent was used to detect cell viability.

7. The improved method for constructing ovarian cancer organoids according to claim 2, characterized in that: The ovarian cancer targeting natural product is prismaticin.

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