Drug-resistant organ model based on interaction of tumor cells and nerve cells as well as preparation and application of drug-resistant organ model

Abstract

The invention discloses preparation and application of a drug-resistant organ model based on interaction of tumor cells and nerve cells, and the preparation method comprises the following steps: mixing tumor cells, PC12 induced and differentiated nerve cells and matrigel, using biocompatible fluorine oil as a continuous phase, preparing microspheres through a microfluidic microdroplet wrapping technology, curing, and culturing to obtain the drug-resistant organ model based on interaction of tumor cells and nerve cells. And obtaining the tumor drug-resistant organ. The organoid has drug resistance to platinum drugs such as cis-platinum, can simulate the drug resistance process mediated by interaction of in-vivo tumors and nerve cells, is used for tumor drug resistance mechanism research, drug resistance reversal drug screening and preclinical drug resistance detection, is easy and convenient to operate and good in uniformity, and has important biological medicine application value.

Description

A drug-resistant organoid model based on tumor cell-neuron cell interaction and its preparation and application Technical Field

[0001] This invention relates to the fields of biotechnology and biomedicine, and more specifically, to a drug-resistant organoid model based on the interaction between tumor cells and nerve cells, and its preparation and application. Background Technology

[0002] Colorectal cancer and liver cancer are among the leading causes of cancer-related morbidity and mortality worldwide. Colorectal cancer, as one of the most common digestive tract tumors, often undergoes a long process from adenoma to carcinoma, closely related to factors such as genetics, diet, and gut microbiota. Liver cancer mainly includes hepatocellular carcinoma and cholangiocarcinoma, with hepatocellular carcinoma being the predominant type. Its occurrence is closely associated with underlying liver diseases such as chronic viral hepatitis (hepatitis B and C), cirrhosis, alcoholic liver disease, and metabolic-related fatty liver disease. Despite significant advancements in surgical resection, local ablation, molecular targeted therapy, and immunotherapy, chemotherapy resistance remains a core challenge leading to treatment failure and tumor recurrence in patients with advanced-stage cancer. The inherent heterogeneity of tumor cells and their dynamic interaction with the complex tumor microenvironment (TME) are the fundamental drivers of drug resistance and disease progression.

[0003] Nerve innervation is a crucial element in the tumor microenvironment of the liver and colon. Under physiological conditions, both the liver and intestines are richly innervated by autonomic and sensory nerves, which finely regulate organ blood flow, metabolism, secretion, and immune balance through the release of neurotransmitters. For example, the sympathetic nervous system can influence hepatic glycogenolysis and immune cell function through the β-adrenergic receptor signaling pathway; the enteric nervous system is the core of independently regulating intestinal motility, secretion, and local reflexes. Under pathological conditions, tumor tissue can induce neurogenesis, forming a microenvironment rich in nerve fibers. These infiltrating nerve cells establish direct neuro-cancer cell synaptic connections or parasecrete neurotransmitters. On the one hand, certain neurotransmitters (such as catecholamines) can significantly promote tumor cell proliferation, invasion, and metastasis by activating specific signaling pathways within cancer cells (such as cAMP / PKA and MAPK); on the other hand, neurotransmitters can also reshape the immune microenvironment and inhibit anti-tumor immune responses. Therefore, nerve cells have become a new and powerful regulator in the tumor microenvironment, and their mechanisms of action await systematic analysis.

[0004] The role of nerve cells in tumor drug resistance is increasingly becoming a research hotspot. Preliminary evidence suggests that neurotransmitter signaling pathways can significantly affect the sensitivity of tumor cells to chemotherapeutic drugs. For example, in pancreatic and prostate cancer, activation of β-adrenergic receptors can mediate resistance to gemcitabine and docetaxel chemotherapy by promoting cell survival signals (such as the AKT pathway) and inhibiting apoptosis. In breast cancer, neuropeptides released by sensory nerves (such as substance P) have also been shown to enhance tumor cell stemness and lead to drug resistance.

[0005] Current research on drug resistance in liver and colon cancer is mainly based on cell and animal models. Traditional 2D cell models lack in vivo multi-cell interactions. Tumor organoids have significant advantages over traditional 2D monolayer cultures in drug screening, primarily in their ability to more realistically simulate the complex structure and function of tumors in vivo. Their three-dimensional structure reconstructs the interactions between cells and between cells and the matrix, forming a gradient microenvironment similar to that of solid tumors (such as the distribution of oxygen, nutrients, and metabolites). This better simulates key physiological and pathological features within the tumor, such as proliferative heterogeneity, drug penetration barriers, and hypoxic cores. This allows organoid-based drug efficacy assessments (especially for chemotherapy and targeted drugs) to more accurately predict in vivo efficacy, significantly reducing false positive or false negative results caused by the oversimplification of the microenvironment in traditional 2D cultures. They are particularly suitable for assessing complex phenotypes such as drug penetration, tumor stem cell activity, and drug resistance development, providing a platform for precision medicine. Furthermore, in mechanistic studies, they can more reliably reveal the impact of drugs on tumor spatial structure, cell signaling pathways, and microenvironment interactions, ultimately improving the success rate of clinical translation of new drug development. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the primary objective of this invention is to provide a method for preparing and applying drug-resistant organoids based on the interaction between tumor cells and nerve cells. This invention uses a matrix gel to mix liver cancer cells or colon cancer cells with nerve cells, and then utilizes microfluidic microdroplet encapsulation technology to prepare high-fidelity organoids that mimic the tumor cell microenvironment, effectively simulating the in vivo tumor drug-resistant environment. The drug-resistant organoids of this invention, representing the interaction between liver cancer cells, colon cancer cells, and nerve cells, possess characteristics of both tumor cells and nerve cells, exhibiting resistance to chemotherapeutic drugs. They can be used for researching the mechanisms by which nerve cells regulate tumor cell drug resistance and for drug development to reverse drug resistance.

[0007] To achieve the above objectives, the present invention provides the following technical solution: The present invention provides a method for preparing tumor drug-resistant organoids, the method comprising: S1) mixing tumor cells with PC12 cell-induced differentiated neural cells and matrix gel to obtain a homogenate; S2) using the homogenate as the dispersed phase and an oil phase reagent as the continuous phase, using microfluidic microdroplet encapsulation technology, controlling the flow rates of the dispersed and continuous phases through a three-way connector, preparing the homogenate into microspheres, and solidifying the obtained microspheres at 35-39℃, obtaining tumor organoid precursors after solidification; S3) suspending the tumor organoid precursors in a complete culture medium to obtain organoids with tumor and neural cell characteristics, i.e., drug-resistant tumor organoids; wherein the tumor cells are one or more of liver cancer cells, colon cancer cells, gastric cancer cells, lung cancer cells, and breast cancer cells.

[0008] Furthermore, the ratio of tumor cells to nerve cells is 1:0.8-1.2, preferably 1:1.

[0009] Furthermore, the PC12-induced differentiated neural cells are obtained by seeding PC12 cells onto a carrier pretreated with poly-L-lysine or collagen, culturing them in a low-serum medium, adding β-nerve growth factor (β-NGF) to induce differentiation, and obtaining mature neural cells after 4-6 days of culture.

[0010] Furthermore, the serum concentration in the low-serum culture medium is 0.5%-2% (volume percentage), preferably 1% horse serum; the concentration of β-NGF added is 80-120 ng / mL, preferably 100 ng / mL; the culture medium is changed every 1-3 days, preferably every 2 days.

[0011] Furthermore, the liver cancer cells are one or more of H22, HepG2, Hep3B, and SMMC-7721, preferably H22.

[0012] Furthermore, the colon cancer cells are one or more of CT26, HT-29, HCT116, and SW480.

[0013] Furthermore, the matrix adhesive is selected from Corning Matrigel™ high-concentration basement membrane matrix adhesive, product number #352434.

[0014] Furthermore, the cell concentration in the matrix gel is (0.8-1.2) × 10⁻⁶ cells per 1 μL of matrix gel. 4 Cells, preferably 1×10⁶ per 1 μL of matrix gel. 4 Each cell.

[0015] Further, the mixing described in step S1 is as follows: after mixing tumor cells, nerve cells and matrix gel, centrifuge at 250-350 g at room temperature for 2-4 minutes, discard the supernatant, add matrix gel according to the cell count results, and gently mix on ice until there are no cell clumps and air bubbles.

[0016] Furthermore, the tumor cells and nerve cells each contain different fluorescent genes.

[0017] Furthermore, the three-way connector described in step S2 is made of polydimethylsiloxane (PDMS). The preparation process is as follows: the PDMS prepolymer is mixed with a curing agent, and after curing, it is cut into a cuboid and a T-shaped channel is created inside.

[0018] Furthermore, the inner diameter of the aforementioned T-shaped channel is 0.8-1.2 mm, preferably a circular inner diameter.

[0019] Furthermore, the microfluidic droplet encapsulation technology involves using a microfluidic device to co-flow the dispersed phase and the continuous phase within a channel, forming uniformly sized droplets and solidifying them to obtain microspheres.

[0020] Furthermore, the operating parameters of the microfluidic droplet encapsulation technology described in step S2 are: the dispersed phase flow rate is 5-8 μL / min, and the continuous phase flow rate is 100-110 μL / min.

[0021] Furthermore, the pretreatment of the microfluidic device in step S2 includes: precooling the oil phase and the device loading the oil phase to 37°C, preferably below 10°C; after the aqueous phase is set up, adjusting the injection pump to fill the pipeline with the oil phase and intersecting it with the aqueous phase in the pipeline, cutting the aqueous phase into microspheres.

[0022] Furthermore, the pretreatment of the microfluidic device in step S2 includes: pre-cooling the 100 μL syringe tip at -20°C, pre-cooling the 1 mL syringe containing the oil phase reagent on ice or at -20°C for more than 30 minutes, connecting the 10 mL syringe to the needle and polytetrafluoroethylene (PTFE) tubing after drawing the oil phase reagent, and adjusting the injection pump to fill the tubing with the oil phase.

[0023] Further, the complete culture medium in step S3 is based on DMEM, DMEM / F12 or RPMI-1640, with 5%-15% fetal bovine serum (by weight), preferably 10% fetal bovine serum.

[0024] Furthermore, the flow rate of the homogenate is 5 μL / min - 8 μL / min, and the flow rate of the oil phase reagent is 100 μL / min - 110 μL / min.

[0025] Furthermore, the oil phase reagent is a biocompatible fluorinated organic solvent. More specifically, the biocompatible fluorinated organic solvent is selected from one or more of hydrofluoroether (HFE) fluorinated oils, perfluoropolyether oils (PFPE), and perfluoronaphthalenes; for example, heptafluoropropyl methyl ether. Preferably, the hydrofluoroether (HFE) fluorinated oil is a 3M Novec™ series fluorinated oil, more preferably a hydrofluoroether fluorinated oil with catalog number HFE7000.

[0026] Furthermore, the curing conditions for the tumor organoid precursor are: curing at 37°C and 5% CO2 for 15-30 minutes.

[0027] Furthermore, the organoid culture medium is based on DMEM, DMEM / F12 or RPMI-1640, with 5%-15% fetal bovine serum added by weight, preferably 10% fetal bovine serum.

[0028] Furthermore, the suspension culture conditions are as follows: cultured at 37°C and 5% CO2 for 7 days, with the organoid culture medium being replaced every 2 days.

[0029] This invention provides a tumor-resistant organoid, which is prepared by the above-described preparation method.

[0030] The present invention also provides the application of the aforementioned tumor drug-resistant organoids in the study of the mechanism by which nerve cells regulate tumor cell drug resistance.

[0031] The present invention also provides the application of the aforementioned tumor drug-resistant organoids in screening drugs for the treatment of tumor drug resistance.

[0032] Furthermore, the tumors targeted in the tumor drug resistance are liver cancer, colon cancer, stomach cancer, lung cancer, and breast cancer.

[0033] Furthermore, the resistance to tumor drugs refers to resistance to antitumor drugs, and the antitumor drugs are preferably platinum-based antitumor drugs. The platinum-based antitumor drugs are selected from cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, cetplatin, and miplatin, among which carboplatin, oxaliplatin, and nedaplatin are preferred.

[0034] The present invention also provides the application of the aforementioned tumor drug-resistant organoids in the preparation of preclinical drug resistance detection models.

[0035] The organoid provided by this invention is a drug-resistant organoid with tumor and nerve cell characteristics. Immunofluorescence results from embodiments of this invention show that the organoid possesses tumor cell characteristics, highly expressing CEA, characteristic of cancer cells; immunofluorescence also shows that the organoid microspheres express the nerve cell marker β-Tubulin III, indicating that tumor cells and nerve cells coexist in the organoid microspheres. The organoid model constructed by this invention possesses both cancerous characteristics and nerve cell features.

[0036] The present invention provides drug-resistant organoids for liver and colon cancer, IC 50 The results showed that, under treatment with a series of concentrations of the anticancer targeted therapy drug cisplatin, the IC50 of organoids co-encapsulating nerve cells in liver cancer and colon cancer was significantly reduced. 50 The drug resistance of the organoids is significantly higher than that of organoids that only encapsulate cancer cells, demonstrating that the interaction model of liver cancer and colon cancer cells with nerve cells in this invention has drug resistance characteristics. Based on this model, not only can we study the drug resistance mechanism of liver cancer and colon cancer, but we can also screen out effective drugs to reverse drug resistance in liver cancer and colon cancer, which has great application value in targeted therapy of liver cancer and colon cancer.

[0037] The beneficial effects of the present invention are as follows: (1) The spheres formed by the traditional low-adsorption spheroidization method have significant intra-batch and inter-batch heterogeneity in size, cell number and structure, resulting in poor experimental reproducibility and inability to accurately simulate the dense microenvironment and cell-matrix interaction of solid tumors, making it difficult to achieve standardization and high throughput. The present invention provides a liver cancer and colon cancer organoid with immunosuppressive neural cell microenvironment characteristics. Based on microfluidic linkage 3D printing technology, liver cancer cells, colon cancer cells and neural cells are co-encapsulated and manufactured in a high-throughput manner to produce uniform and controllable tumor drug-resistant organoid precursors. After in vitro culture and maturation, liver cancer and colon cancer organoids with immunosuppressive neural cell microenvironment characteristics are obtained.

[0038] (2) The organoids for liver cancer and colon cancer provided by this invention have drug resistance and can be used to study the drug resistance mechanism of tumors such as liver cancer and colon cancer, as well as to screen for drugs that reverse drug resistance in liver cancer and colon cancer through high-throughput screening. This will help to develop new targeted drugs to improve the immunotherapy effect of liver cancer and colon cancer. It can be widely used in the fields of medicine and biology and generate huge social and economic benefits. Attached Figure Description

[0039] Figure 1: Construction of drug-resistant organoids from liver cancer and colon cancer cells interacting with nerve cells. Includes a construction flowchart (A); bright-field images of liver cancer and colon cancer organoid cultures (Day 1, Day 2, Day 3, Day 4), scale bar: 50 μm (B); HE staining identification of liver cancer and colon cancer organoids (C), scale bar: 100 μm; and a size chart of liver cancer and colon cancer organoids (D).

[0040] Figure 2: Detection of tumor marker expression in organoids of liver cancer and colon cancer. Immunofluorescence results of organoids, including organoids containing liver cancer cells and nerve cells, and organoids containing colon cancer cells and nerve cells. Fluorescence images of CEA, a marker of liver cancer cells, and β-Tubulin III, expressed on Day 4, and β-Tubulin III, a marker of nerve cells, expressed on Day 4, scale bar: 500 μm (A); Immunofluorescence statistical graph of CEA and β-Tubulin III, markers of liver cancer cells, expressed on Day 4, and β-Tubulin III, markers of nerve cells, expressed on Day 4, scale bar: 500 μm (B); Immunofluorescence statistical graph of CEA and β-Tubulin III, markers of colon cancer cells, expressed on Day 4 (C).

[0041] Figure 3: Validation of organoid drug resistance. IC50 after drug treatment. 50 The figures show samples including organoids that co-encapsulate nerve cells and liver cancer cells, and organoids that co-encapsulate liver cancer cells (A); organoids that co-encapsulate nerve cells and colon cancer cells, and organoids that co-encapsulate colon cancer cells (B).

[0042] Figure 4: Validation of organoid drug resistance. Fluorescence images of CEA (a cancer cell marker) and β-Tubulin III (a neuronal cell marker) in liver cancer organoids after administration of 2 μM, 4 μM, and 8 μM cisplatin, Scale bar: 500 μm (A). Fluorescence images of CEA and β-Tubulin III (a cancer cell marker) in colon cancer organoids after administration of 4.5 μM, 9 μM, and 18 μM cisplatin, Scale bar: 500 μm (B).

[0043] Figure 5: Fluorescence statistics of CEA, a cancer cell marker, and β-Tubulin III, a neuronal cell marker, in liver cancer organoids (A); Fluorescence statistics of CEA, a cancer cell marker, and β-Tubulin III, a neuronal cell marker, in colon cancer organoids (B). Detailed Implementation

[0044] To better understand the present invention, it is now further described with reference to the following embodiments and accompanying drawings. The embodiments are for illustrative purposes only and do not limit the invention in any way. In the embodiments, all original reagents and materials are commercially available, and experimental methods not specifically specified are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0045] Example 1: Construction of organoids for interaction between liver cancer, colon cancer cells and nerve cells (1) Preparation of liver cancer organoids by microfluidic linkage 3D printing technology PC12 cells were printed at 1×10 4The cells were seeded in 6-well plates pretreated with poly-L-lysine or collagen and cultured in a medium containing low serum (1% horse serum). Subsequently, 100 ng / mL of β-NGF was added to the medium, and the medium was changed every two days. Differentiated and mature neural cells were obtained after 5 days of culture.

[0046] T-connectors were fabricated using polydimethylsiloxane (PDMS). The PDMS prepolymer and curing agent were mixed at a mass ratio of 10:1, and air bubbles were removed. The mixture was cured in a 70°C oven for 2 hours to form a PDMS elastomer.

[0047] The PDMS block was cut into cuboids, and a T-shaped tee connector was created using a 1mm wide biopsy piercing tool.

[0048] One day in advance, pre-cool the sterilized 100 μL pipette tips at -20°C. 30 minutes in advance, pre-cool the 1 mL syringe containing 0.5 mL of oil phase on ice or at -20°C. Draw 5 mL of oil phase into a 10 mL syringe, connect the needle and approximately 20 cm of PTFE tubing to the right end of the tee box, and connect approximately 10 m of PTFE tubing to the left end of the tee box. Connect the syringe to an external syringe pump (flow rate: 110 μL / min). Adjust the syringe pump to fill the 20 cm PTFE tubing connected to the needle with the oil phase from the 10 mL syringe. Transfer the H22 liver cancer cells, nerve cells, and the matrix gel mixture to a 1.5 mL centrifuge tube, centrifuge at 300 g for 3 min at room temperature, discard the supernatant, and, based on the cell count, use a 1×10⁻⁶ ppm solution. 4Calculate the required matrix gel volume by adding 1 μL of matrix gel to each cell. Add matrix gel to the cell pellet using a pre-chilled pipette tip, gently stir several times on ice, and then pipette to ensure no air bubbles are generated. Mix until no cell clumps remain. Inject the cell-matrix mixture into a pre-chilled 1 mL syringe containing the oil phase, attach a needle and approximately 10 cm of PTFE tubing, and then connect it to a syringe pump (flow rate: 15 μL / min) inside the refrigerator, attaching it to the center hole of a three-way valve. Adjust the syringe pump so that the cell-matrix mixture appears in the 10 cm PTFE tubing. First start the external syringe pump, then start the internal syringe pump. Shear the cell-matrix mixture into monodisperse droplets through the three-way valve, and then transfer them to the approximately 10 cm PTFE tubing. Stop both syringe pumps when the PTFE tubing connected to the 1 mL syringe no longer contains cell-matrix mixture. Place the PTFE tubing carrying the cell-matrix mixture droplets in a 37°C incubator for 30 min to fix, thus obtaining cell-matrix microspheres. Using a 1 mL syringe, matrix gel microspheres were blown into a petri dish containing human lung organoid culture medium. The oil phase on the surface of the petri dish was gently aspirated using a 1 mL syringe with a needle, resulting in matrix gel microspheres of uniform size and cell homogeneity. The obtained microspheres were then incubated at 37°C for 25 minutes to solidify and obtain precursors for liver cancer and colon cancer organoids.

[0049] (2) Culture of liver cancer and colon cancer organoids: The liver cancer and colon cancer organoid precursors obtained in step (1) were cultured in suspension in a prepared complete culture medium. The complete culture medium was DMEM as the basal medium, containing 10% (by weight) fetal bovine serum. The culture was carried out at 37°C and 5% CO2 for 7 days, and the complete culture medium was replaced every 2 days. After in vitro culture and maturation, liver cancer organoids with characteristics of liver cancer and colon cancer were obtained.

[0050] (3) Results: Figure 1A shows the flowchart of the construction of organoids for liver cancer and colon cancer, introducing the preparation and application of these organoids; Figure 1B shows the bright field diagrams of the growth of the two groups of organoids over the culture time (Day 1, Day 2, Day 3, Day 4). In the complete culture medium, the cells of the two groups of organoids began to proliferate and differentiate, and the structure gradually grew as the number of days increased; Figure 1C shows the HE staining images of the organoids, and the results show that the organoids for liver cancer and colon cancer have the pathological characteristics of liver cancer and colon cancer. Figure 1D shows the changes in the diameter of the two types of organoids for liver cancer and colon cancer over the growth time.

[0051] Example 2: Characterization of organoids of liver cancer and colon cancer (1) Immunofluorescence detection The mature liver cancer organoids obtained at the end of the first stage of Example 1 were subjected to immunofluorescence staining of paraffin sections.

[0052] 1.1 First, the organoid microspheres were embedded in paraffin. The specific steps are as follows: Fixation: The organoid microspheres were fixed with 4% paraformaldehyde; Dehydration: Dehydration was then carried out sequentially with a gradient of alcohols (85%, 95%, 95%, 100%, 100% alcohol); Clearing: After dehydration, xylene was added for clearing; Paraffin impregnation: The paraffin solution was gently and slowly dripped onto the organoids and placed in an incubator for 30 minutes; Embedding: After paraffin impregnation, the embedding box was covered and placed in a freezer to allow the paraffin solution to solidify.

[0053] 1.2 Then, the paraffin blocks were sectioned. The specific steps are as follows: After mounting the organoid paraffin blocks on the paraffin microtome, the sections were trimmed and then cut into 3μm sections. After spreading, the sections were baked for 10 minutes.

[0054] 1.3 Finally, the paraffin sections were stained with immunofluorescence. The specific steps are as follows: Tissue rehydration: Xylene I for 10 minutes, Xylene II for 10 minutes, Anhydrous ethanol I for 10 minutes, Anhydrous ethanol II for 10 minutes, 95% ethanol I for 10 minutes, 95% ethanol II for 10 minutes, and rinse with pure water for 1 minute; Antigen retrieval: Place the slide containing the tissue section in 1× EDTA (pH 9.0) antigen retrieval solution, place it in a microwave oven, heat on medium-high for 4 minutes until boiling, heat on bottom for 20 minutes, cool to room temperature, and wash 3 times with PBS for 3 minutes each time; Blocking: Draw a circle on the front of the slide at the location of the organoid using an immunohistochemical pen, add blocking solution (5% BSA + 0.1% Triton X-100) to the circle of the water-resistance pen, and block at room temperature for 1 hour; Primary antibody staining: Add the primary antibody, diluted with blocking solution according to the recommended concentration in the instructions, place the slide in a humidified chamber, and incubate overnight at 4°C; Secondary antibody staining: Incubate with the corresponding secondary antibody, diluted according to the recommended concentration in the instructions, and incubate at room temperature in the dark for 1 hour. Hours; DAPI staining: Add DAPI containing anti-fluorescence quenching mounting medium to a glass slide and mount; Fluorescence photography: After mounting, take a picture using a fluorescence microscope.

[0055] (2) Hematoxylin-eosin staining (HE) staining of organoid microspheres: paraffin embedding and sectioning according to steps (1) ① and ② of Example 3.

[0056] HE staining steps are as follows: Dewaxing: Immerse the sections in xylene I for 5 minutes, xylene II for 5 minutes, anhydrous ethanol I for 3 minutes, anhydrous ethanol II for 3 minutes, 95% ethanol for 3 minutes, and then soak in tap water for about 10 seconds; Hematoxylin staining of cell nuclei: Immerse the sections in hematoxylin staining for 7 minutes, soak and rinse in tap water for 10 seconds, differentiate in 1% acidic ethanol differentiation solution for 3-5 seconds, soak and rinse in tap water for 10 seconds, return to blue with 1% ammonia solution for 3-5 seconds, and return to blue with running tap water for 5 minutes; Eosin staining of cytoplasm: Immerse the sections in eosin staining solution for 3-5 seconds, and then soak the sections in tap water for 5 minutes; Dehydration and mounting: Immerse the sections in 95% ethanol for 1 minute, anhydrous ethanol I for 1 minute, anhydrous ethanol II for 1 minute, and xylene for 3 minutes to dehydrate and clear, and then mount with neutral resin.

[0057] (3) Results: Figures 2A and 2B show that both organoids expressed CEA (a cancer cell marker) and β-Tubulin III (a neuronal cell marker), indicating that the liver cancer cells (H22) and colon cancer cells (CT26) maintained their own tumor cell biological characteristics and did not lose their core cancer cell phenotype due to co-culture or microfluidic preparation. At the same time, it shows that the neuronal cells (PC12) survived in the co-culture system and maintained their own neuronal cell functional phenotype, and were not inhibited or induced to differentiate into other cell types by tumor cells.

[0058] The results showed that both types of organoids possessed characteristics of both cancer cells and nerve cells. The organoids successfully simulated the microenvironment in which tumor cells and nerve cells coexist in vivo, laying the foundation for subsequent research on the mechanisms by which nerve cells regulate tumor drug resistance and for drug screening.

[0059] Example 3: Drug Resistance Detection of Organoids from Liver Cancer and Colon Cancer (1) Cisplatin Treatment Cisplatin was prepared by diluting the drug in organoid complete culture medium to a series of working concentrations from low to high: 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, and 50 μM. Organoids were cultured for 5 days. Five organoid microspheres were added to each well of a 96-well opaque plate, and 100 μL of different working concentrations of cisplatin-containing culture medium were added to each well. Three parallel replicates were set up, with organoid microspheres in complete culture medium without the drug serving as the control group. Organoids were treated with the drug for 24 h.

[0060] (2) Cell viability detection after drug treatment: CellTiter-Glo® 3D Cell Viability Assay (Promega) luminescent cell viability assay kit was used to detect the cell viability of organoids after drug treatment. This kit is a homogeneous and rapid assay method that detects the number of viable cells in the culture by quantitatively measuring ATP. The Cell Viability Assay reagent was thawed overnight at 4°C, and the reagent was equilibrated in a water bath at 22°C for 30 minutes and mixed well. The 96-well light-proof plate containing organoids was taken out of the 37°C incubator, and 100 μL of reagent equal to the volume of culture medium was added to each well. The plate was shaken on the microplate reader for 5 minutes, and then incubated for 25 minutes before reading was taken.

[0061] (3) Results Description Figure 3A shows the IC50 of organoid treatment for liver cancer. 50 Figure. IC50 of hepatocellular carcinoma organoid microspheres with an immunosuppressive neuronal microenvironment after treatment with a range of concentrations of the targeted therapeutic drug cisplatin. 50 The concentration was 36 μM, while the organoid IC without nerve cells was... 50 The concentration was 8 μM, demonstrating that the organoids constructed by this method, which involve interaction between liver cancer cells and nerve cells, possess an immunosuppressive microenvironment that can reflect the drug resistance characteristics of liver cancer in clinical practice. Based on this model, effective drugs to reverse drug resistance in liver cancer can be screened, which has great application value in targeted therapy for liver cancer.

[0062] Figure 3B illustrates the IC of organoid drug treatment for colon cancer. 50 Figure. IC50 of colon cancer organoid microspheres with an immunosuppressive neuronal microenvironment after treatment with a range of concentrations of the targeted therapeutic drug cisplatin. 50 The concentration was 37 μM, while the organoid IC without nerve cells was... 50 It is 18 μM.

[0063] Figure 4A shows the expression of the cancer marker CEA and the neuronal marker β-Tubulin III in liver cancer organoids at different cisplatin concentrations. Figure 4B shows the expression of the cancer marker CEA and the neuronal marker β-Tubulin III in colon cancer organoids at different cisplatin concentrations.

[0064] Figures 5A and 5B show the statistical distribution of the cancer marker CEA and the neuronal marker β-Tubulin III in liver cancer organoids and colon cancer organoids at different cisplatin concentrations.

[0065] Figures 4 and 5 show that, regarding the expression of the cancer cell marker CEA, the control group, containing only tumor cells, exhibited a significant decrease in fluorescence intensity with increasing cisplatin concentration (e.g., from 0 to 8 μmol in liver cancer organoids), indicating that cancer cells were inhibited by the drug and underwent apoptosis. The experimental group, containing both tumor cells and nerve cells, showed a much smaller decrease in CEA fluorescence intensity with increasing cisplatin concentration compared to the control group. For example, in Figure 5A, after treatment with 8 μmol cisplatin, the experimental group maintained high CEA expression, indicating that the presence of nerve cells protected cancer cells, enabling them to survive even in high-concentration drug environments, further validating the drug resistance characteristic. Regarding the expression of the nerve cell marker β-Tubulin III, in the experimental group, even under high cisplatin treatment (e.g., 8 μmol for liver cancer and 18 μmol for colon cancer), this marker remained stably expressed, as shown in the Merge plots of Figures 4A and 4B. This indicates that nerve cells themselves are insensitive to cisplatin and can continuously survive and function in the drug environment, constructing an immunosuppressive microenvironment. This result rules out the possibility that drug resistance is caused by mutations in the tumor cells themselves, and clarifies that the persistent presence of nerve cells maintains the microenvironment and protects the tumor cells. It demonstrates that the organoids of colorectal cancer cells interacting with nerve cells constructed using this method possess an immunosuppressive microenvironment that can reflect the drug resistance characteristics of colorectal cancer in clinical practice. Based on this model, it is possible to screen for specific drugs that reverse drug resistance in colorectal cancer, which has significant application value in targeted therapy for colorectal cancer.

[0066] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing tumor-resistant organoids, characterized in that, The preparation method includes: S1) mixing tumor cells with PC12 cell-induced differentiated neural cells and matrix gel to obtain a homogenate; S2) using the homogenate as the dispersed phase and an oil phase reagent as the continuous phase, using microfluidic microdroplet encapsulation technology, controlling the flow rates of the dispersed and continuous phases through a three-way connector, preparing the homogenate into microspheres, and solidifying the obtained microspheres at 35-39℃ to obtain tumor organoid precursors; S3) suspending the tumor organoid precursors in a complete culture medium to obtain organoids with tumor and neural cell characteristics, i.e., drug-resistant tumor organoids; wherein, the tumor cells are one or more of liver cancer cells, colon cancer cells, gastric cancer cells, lung cancer cells, and breast cancer cells.

2. The preparation method according to claim 1, characterized in that, The ratio of tumor cells to nerve cells is 1:0.8-1.

2. Preferably, the PC12-induced differentiated nerve cells are obtained by: seeding PC12 cells onto a carrier pretreated with poly-L-lysine or collagen, culturing in a low-serum medium, adding β-nerve growth factor to induce differentiation, and obtaining mature nerve cells after 4-6 days of culture. Preferably, the serum concentration in the low-serum medium is 0.5%-2% by volume, and the concentration of β-NGF added is 80-120 ng / mL. The medium is changed every 1-3 days. Preferably, the cell concentration in the matrix gel is (0.8-1.2) × 10⁻¹¹ cells per μL of matrix gel. 4 Each cell.

3. The preparation method according to claim 1, characterized in that, The tee connector described in step S2 is prepared using polydimethylsiloxane. The preparation process involves mixing PDMS prepolymer with a curing agent, curing it, cutting it into a cuboid, and then creating a T-shaped channel inside. Preferably, the internal diameter of the T-shaped channel is 0.8-1.2 mm; preferably, the tee connector has a circular inner diameter.

4. The preparation method according to claim 1, characterized in that, The microfluidic droplet encapsulation technology involves using a microfluidic device to co-flow a dispersed phase and a continuous phase within a channel, forming uniformly sized droplets and solidifying them to obtain microspheres. Preferably, the operating parameters of the microfluidic droplet encapsulation technology in step S2 are: a dispersed phase flow rate of 5-8 μL / min and a continuous phase flow rate of 100-110 μL / min.

5. The preparation method according to claim 1, characterized in that, Furthermore, the oil phase reagent is a biocompatible fluorinated organic solvent, and even further, the biocompatible fluorinated organic solvent is selected from one or more of hydrofluoroether (HFE) fluorinated oils, perfluoropolyether oils (PFPE), and perfluoronaphthalenes; preferably, the flow rate of the mixture is 5 μL / min - 8 μL / min, and the flow rate of the oil phase reagent is 100 μL / min - 110 μL / min.

6. The preparation method according to claim 1, characterized in that, The complete culture medium in step S3 is based on DMEM, DMEM / F12 or RPMI-1640, with 5%-15% fetal bovine serum added by weight; preferably, the organoid culture medium is based on DMEM, DMEM / F12 or RPMI-1640, with 5%-15% fetal bovine serum added by weight; preferably, the suspension culture conditions are: cultured at 37°C and 5% CO2 for 7 days, with the organoid culture medium being replaced every 2 days.

7. A tumor-resistant organoid, characterized in that, It is prepared by the preparation method described in any one of claims 1-6.

8. The application of the tumor drug-resistant organoids as described in claim 7 in the study of the mechanism by which nerve cells regulate tumor cell drug resistance.

9. The application of the tumor drug-resistant organoids of claim 7 in screening drugs for treating tumor drug resistance; preferably, the tumors targeted in the tumor drug resistance are liver cancer, colon cancer, gastric cancer, lung cancer, and breast cancer; preferably, the drug resistance in the tumor drug resistance is resistance to antitumor drugs, and the antitumor drugs are more preferably platinum-based antitumor drugs, wherein the platinum-based antitumor drugs are selected from cisplatin, carboplatin, oxaliplatin, nedaplatin, lobaplatin, cetaplatin, and miplatin, wherein carboplatin, oxaliplatin, and nedaplatin are preferred.

10. The application of the tumor drug-resistant organoids according to claim 7 in the preparation of preclinical drug resistance detection models.

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