Cellular drugs and their efficacy evaluation methods and applications in bladder cancer treatment

Abstract

This invention belongs to the field of biomedical technology, specifically relating to cell-based drugs and their efficacy evaluation methods and applications in bladder cancer treatment. This invention involves adding sensitized PBMC suspension to organoid suspensions for co-culture. After co-culture, cells are collected to obtain the cell-based drug. Based on this cell-based drug, a bladder cancer efficacy evaluation model is constructed: co-culturing the cell-based drug with bladder cancer organoids overcomes the shortcomings of traditional models in simulating tumor-immune interactions. The efficacy evaluation results have high clinical relevance, and the cell-based drug exhibits strong targeted killing ability. This model is suitable for developing individualized treatment plans for bladder cancer and high-throughput screening of anti-bladder cancer drugs, demonstrating significant clinical translational value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to cell-based drugs and their efficacy evaluation methods and applications in bladder cancer drugs. Background Technology

[0002] Bladder cancer is one of the most common malignant tumors of the urinary system worldwide. Current treatment for bladder cancer is based on surgical resection, combined with chemotherapy (such as gemcitabine + cisplatin), targeted therapy (such as erdatinib), or immune checkpoint inhibitors (such as pembrolizumab). However, there are significant individual differences in clinical efficacy, and there is a lack of an effective in vitro pharmacodynamic evaluation system to predict treatment effects, resulting in frequent ineffective treatments. This not only delays the best treatment time for patients, but also increases the risk of drug toxicity and side effects and medical costs.

[0003] Traditional methods for evaluating the efficacy of bladder cancer drugs primarily rely on two types of models: animal models and two-dimensional cell culture models. Animal models cannot simulate the complex tumor microenvironment in the human body. These models require transplanting bladder cancer cell lines or patient tumor tissue into immunodeficient mice. While tumor growth trends can be observed, the immune systems and metabolic pathways of mice differ fundamentally from those of humans, particularly in their inability to replicate tumor-immune cell interactions. This results in a high deviation rate between the efficacy of immunotherapy drugs in animal models and clinical reality, making them unsuitable as a basis for clinical drug use. Furthermore, animal models have a long construction cycle of 4-8 weeks, leading to high costs and failing to meet the need for rapid matching of treatment plans in personalized medicine, let alone achieving high-throughput drug screening. Two-dimensional cell culture models differ significantly from tumors in patients. These models are obtained through long-term in vitro passages, gradually losing the histological characteristics and molecular phenotypes of the primary tumor during culture, resulting in significant discrepancies between the drug sensitivity of the cell lines and those of patient tumor cells. Simultaneously, the two-dimensional culture environment only provides planar growth space, lacking the matrix microenvironment required for tumor cell growth, further leading to a disconnect between cell biological behavior and the in vivo state.

[0004] The evaluation capabilities of traditional models have lagged far behind the innovations in bladder cancer treatment technologies, especially with the rise of cell immunotherapy (such as CAR-T cell therapy and tumor-infiltrating lymphocyte therapy). Existing models cannot simulate the targeted killing process of immune cells against tumors, resulting in low efficiency in the research and clinical translation of novel cell drugs. Therefore, constructing a highly efficient and accurate drug efficacy evaluation system that can simulate the human tumor microenvironment, preserve individual tumor characteristics, and address the challenges in clinical treatment of bladder cancer has become a key technological bottleneck for solving these challenges and promoting the development of new drugs. Summary of the Invention

[0005] To address the shortcomings of existing bladder cancer drug efficacy evaluation models, such as their inability to simulate the complex tumor microenvironment in the human body, significant differences from tumors in patients, long construction cycles, high costs, and difficulty in achieving high-throughput screening, this invention provides a cell-based drug and its efficacy evaluation method and application in bladder cancer drugs.

[0006] Terminology Explanation:

[0007] The term “A83-01” as used in this invention refers to an effective inhibitor of TGF-β type I receptors ALK5, ALK4 and ALK7, purchased from TOCRIS, catalog number 233-FB-025.

[0008] The term "recombinant human epidermal growth factor" as used in this invention refers to a small peptide composed of 53 amino acid residues. It is a member of the EGF-like family and a multifunctional growth factor that has a strong pro-mitotic effect on a variety of tissue cells both in vivo and in vitro. CAS: 62253-63-8.

[0009] The term "recombinant human Noggin protein" as used in this invention refers to Noggin, one of the most classic growth factors in organoid culture. Its high activity, batch-to-batch stability, and lack of contamination are key to the success of organoid experiments. Recombinant human Noggin protein is provided in liquid form, is highly active, contains no animal-derived components, and has low endotoxin levels. It is purchased from Nearshore Protein, catalog number CB89.

[0010] The term “recombinant human R-spondin 1” as used in this invention refers to a protein belonging to the R-Spondin family that encodes a secretion-activating protein having two cysteine-rich furin-like domains and a clotting protein type 1 domain. The recombinant human R-spondin 1 used in this invention was purchased from Nearshore Protein, catalog number C-6His.

[0011] The term “recombinant human FGF-basic” as used in this invention refers to recombinant human basic fibroblast growth factor FGFbasic, which belongs to the FGF family. It is a heparin-binding growth factor that can be secreted by endothelial cells, smooth muscle cells, and macrophages. It can promote the proliferation of a range of cells, including mesenchymal cells, neuroectodermal cells, vascular endothelial cells, and smooth muscle cells. It was purchased from R&D, catalog number 233-FB-025.

[0012] The term "recombinant human FGF-10" as used in this invention refers to FGF-10, a member of the fibroblast growth factor family, which is naturally present in the human body and whose core function is to regulate cell proliferation, differentiation, and tissue repair. Recombinant human FGF-10 is a protein-based biological product prepared using genetic engineering technology, with a structure identical to that of natural human FGF-10, and was purchased from R&D (Catalog No. 345-FG-025).

[0013] The technical solution of this invention is as follows:

[0014] On one hand, the present invention provides a cell-based drug, the preparation method of which includes the following steps:

[0015] S1. Bladder cancer organoids were resuspended in co-culture medium to obtain organoid suspensions;

[0016] S2 and PBMC were resuspended in co-culture medium to obtain PBMC suspension. MUC1 peptide was added to make the final concentration of MUC1 peptide in PBMC suspension 5-8 μg / mL. After incubation, sensitized PBMC suspension was obtained.

[0017] S3. According to the cell ratio of bladder cancer organoids to sensitized PBMCs = 1:12-18, add sensitized PBMC suspension to organoid suspension, co-culture, collect cells after co-culture, and obtain cell drug;

[0018] The co-culture medium contains IL-2, IL-12, anti-CD28 antibody and anti-PD-1 antibody.

[0019] Specifically, the co-culture medium contains 15-25 ng / mL IL-2, 5-10 ng / mL IL-12, 1-2 μg / mL anti-CD28 antibody and 7.5-15 μg / mL anti-PD-1 antibody.

[0020] More specifically, the co-culture medium contains 15-16, 16-17, 17-18, 18-19, 19-20, 20-21, 21-22, 22-23, 23-24 or 24-25 ng / mL IL-2.

[0021] Preferably, the co-culture medium contains 20-21, 21-22, 22-23, 23-24 or 24-25 ng / mL IL-2.

[0022] More preferably, the co-culture medium contains 20 ng / mL IL-2.

[0023] More specifically, the co-culture medium contains 5-6, 6-7, 7-8, 8-9, or 9-10 ng / mL IL-12.

[0024] Preferably, the co-culture medium contains 5-6, 6-7, or 7-8 ng / mL IL-12.

[0025] More preferably, the co-culture medium contains 5 ng / mL IL-12.

[0026] More specifically, the co-culture medium contains 1.0-1.1, 1.1-1.2, 1.2-1.3, 1.3-1.4, 1.4-1.5, 1.5-1.6, 1.6-1.7, 1.7-1.8, 1.8-1.9 or 1.9-2.0 μg / mL of anti-CD28 antibody.

[0027] Preferably, the co-culture medium contains 1.0-1.1, 1.1-1.2, 1.2-1.3, 1.3-1.4 or 1.4-1.5 μg / mL of anti-CD28 antibody.

[0028] More preferably, the co-culture medium contains 1.0 μg / mL of anti-CD28 antibody.

[0029] More specifically, the co-culture medium contains 7-8, 8-9, 9-10, 10-11, 11-12, 12-13, 13-14 or 14-15 μg / mL of anti-PD-1 antibody.

[0030] Preferably, the co-culture medium contains 7-8, 8-9, or 9-10 μg / mL of anti-PD-1 antibody.

[0031] More preferably, the co-culture medium contains 10 μg / mL of anti-PD-1 antibody.

[0032] Specifically, the co-culture medium is prepared by mixing organoid culture medium with immunobasal medium, and then adding IL-2, IL-12, anti-CD28 antibody and anti-PD-1 antibody to obtain co-culture medium; the organoid culture medium is used for the culture of bladder cancer organoids; and the immunobasal medium is used for the culture of PBMCs.

[0033] Preferably, the organoid culture medium is a culture medium containing recombinant human epidermal growth factor, recombinant human Noggin, recombinant human R-spondin 1, recombinant human FGF-10, recombinant human FGF-basic and A83-01.

[0034] More preferably, the organoid culture medium is a culture medium containing 25-75 ng / mL recombinant human epidermal growth factor, 75-125 ng / mL recombinant human Noggin, 800-1200 ng / mL recombinant human R-spondin 1, 400-600 ng / mL recombinant human FGF-10, 15-25 ng / mL recombinant human FGF-basic and 3-7 μM A83-01.

[0035] More preferably, the organoid culture medium is: Advanced DMEM / F12 medium containing 50 ng / mL recombinant human epidermal growth factor, 100 ng / mL recombinant human Noggin, 1000 ng / mL recombinant human R-spondin 1, 500 ng / mL recombinant human FGF-10, 20 ng / mL recombinant human FGF-basic and 5 μM A83-01.

[0036] More specifically, the immune basal culture medium includes any one or more of RPMI 1640 medium, IMDM medium, DMEM medium, DMEM / F12 medium, MEM medium, α-MEM medium, and Ham's F-12 medium.

[0037] Preferably, the basic immune culture medium is RPMI 1640 medium.

[0038] Preferably, the volume ratio of the organoid culture medium to the immunobasal culture medium is 1:0.1-10.

[0039] More preferably, the volume ratio of the organoid culture medium to the immunobasal culture medium is 1:1.

[0040] Specifically, the final concentration of the MUC1 peptide in the PBMC suspension in step S2 is 5-6, 6-7, or 7-8 μg / mL.

[0041] Preferably, the final concentration of the MUC1 peptide in the PBMC suspension in step S2 is 5 μg / mL.

[0042] Specifically, the incubation described in step S2 includes incubation at 35-40°C for 24-48 hours.

[0043] Preferably, the incubation in step S2 includes incubation at 37°C and 5% CO2 for 24 hours.

[0044] Specifically, the ratio of bladder cancer organoids to sensitized PBMCs in step S3 is 1:12-13, 13-14, 14-15, 15-16, 16-17, or 17-18.

[0045] Preferably, the ratio of bladder cancer organoids to sensitized PBMCs in step S3 is 1:15.

[0046] Specifically, the co-cultivation mentioned in step S3 includes co-cultivation at 35-40℃ for 15-30 days.

[0047] Preferably, the co-cultivation in step S3 includes co-cultivation at 37°C and 5% CO2 for 15 days.

[0048] On the other hand, the present invention provides a method for preparing the cell drug described in any of the above claims.

[0049] In another aspect, the present invention provides the application of the cell drug described in any of the above claims in the preparation of a bladder cancer drug efficacy evaluation model, wherein the method for preparing the bladder cancer drug efficacy evaluation model is as follows: co-culturing the cell drug with bladder cancer organoids to obtain the bladder cancer drug efficacy evaluation model.

[0050] Preferably, the target-efficacy ratio of the cell drug to the bladder cancer organoid is 5:1.

[0051] In another aspect, the present invention provides a method for evaluating the efficacy of a bladder cancer drug, wherein the method includes using any of the cell drugs or bladder cancer drug efficacy evaluation models described above.

[0052] In another aspect, the present invention provides a method for evaluating drug efficacy, the method comprising:

[0053] (1) A bladder cancer drug efficacy evaluation model was obtained by co-culturing cell drugs with bladder cancer organoids;

[0054] (2) Use a bladder cancer drug treatment efficacy evaluation model to evaluate the anti-bladder cancer efficacy of bladder cancer drugs.

[0055] The beneficial effects of this invention are as follows:

[0056] This invention utilizes the characteristics of bladder cancer organoids that retain the histological features and molecular phenotypes of primary tumors, and combines them with cytotoxic T cells induced from peripheral blood mononuclear cells to construct a co-culture model. This model can simulate the interaction between human tumor and immune cells, solving the problem that traditional animal models and two-dimensional cell culture models cannot simulate the complex tumor microenvironment, and making the efficacy evaluation results closer to clinical practice.

[0057] Cellular drugs (CTLs) induced by a specific concentration of cytokines and antibody combinations, as identified by flow cytometry, exhibited a high proportion of CD3+. + CD8 + The cells and high CD107a positivity rate showed significant targeted killing ability against bladder cancer organoids and could effectively activate their proliferation, providing a new and effective means for the treatment of bladder cancer.

[0058] The efficacy evaluation method of this invention is simple to operate and has a relatively short cycle. It can accurately evaluate the effects of cell drugs alone and in combination with traditional anticancer drugs. It can not only provide a basis for optimizing clinical combination treatment plans, but also meet the evaluation needs of individualized treatment. At the same time, it provides an efficient platform for screening anti-bladder cancer drugs, reduce medical costs, and reduce the risks of ineffective treatment. Attached Figure Description

[0059] Figure 1 Bright-field image of bladder cancer organoid culture; scale bar in the image is 200 μm.

[0060] Figure 2 HE staining images of bladder cancer organoids; the left image shows HE staining of human bladder cancer tissue; the right image shows HE staining of organoids derived from human bladder cancer tissue; the scale bar in the images is 100 μm.

[0061] Figure 3 Immunofluorescence staining image of Ki67 in bladder cancer organoids; scale bar in the image is 100 μm. Detailed Implementation

[0062] The present invention will be further clearly and completely illustrated below through examples. These examples are only a part of the embodiments of the present invention and are not intended to limit the present invention, but are only for illustrating the present invention. Unless otherwise specified, the experimental methods used in the following examples are all routine experiments. The CAS number of the MUC1 peptide used is 149205-73-2; the anti-CD28 antibody was purchased from BioLegend, 302930; and the anti-PD-1 antibody was purchased from R&D Systems, MAB1086. Unless otherwise specified, all materials and reagents used in the following examples can be obtained commercially.

[0063] Basic Example 1: Construction and Identification of Bladder Cancer Organoids

[0064] 1. Preparation of bladder cancer culture medium:

[0065] Advanced DMEM / F12 was used as the basal medium, and recombinant human epidermal growth factor 50 ng / mL, recombinant human Noggin 100 ng / mL, recombinant human R-spondin 1 1000 ng / mL, recombinant human FGF-10 500 ng / mL, recombinant human FGF-basic 20 ng / mL, and A83-01 5 μM were added to obtain bladder cancer culture medium.

[0066] 2. Take a fresh tumor tissue sample from the patient's surgically removed tumor. Wash the sample four times with sterile PBS buffer, 5 minutes each time. Then, cut the washed tissue into pieces approximately 1 mm in size using a scalpel. 3 Transfer tissue samples of various sizes to 15 mL centrifuge tubes and add an appropriate amount of TrypLE. TMThe Express enzyme was placed in a 37°C water bath shaker at 60 rpm for 40 minutes, gently shaking every 5 minutes. After digestion, the digestion process could be observed under a microscope. After digestion was stopped, the sample was pipetted and filtered through a 100-mesh filter to obtain a cell clump suspension. Finally, the suspension was centrifuged at 1500 rpm for 10 minutes in a pre-cooled 4°C centrifuge, and the supernatant was discarded. This process was repeated three times to obtain the cell clump pellet.

[0067] 3. Add the cell pellet to Matrigel ® The matrix (volume ratio 1:2) was used to obtain encapsulated cell clusters, which were then added to bladder cancer culture medium for organoid construction at 37°C and 5% CO2.

[0068] 4. Primary (P0) culture growth process of bladder cancer organoids: On day D0, single cells and small cell clusters are visible; by day D3, distinct 3D cell spheroids are observed; continuing growth to day D5, these cell spheroids significantly increase in diameter and vesicles appear. Growth process of bladder organoids after first passage (P1 generation): Passaged bladder cancer organoids form closed vesicle structures by day D1; by day D3, the vesicle diameter significantly increases; continuing growth to day D5, the diameter continues to increase, allowing for further passage. Figure 1 .

[0069] On day 7 of culture, bladder cancer organoids were collected for subsequent experiments. HE staining showed ( Figure 2 The arrangement of cells, nuclei, cytoplasmic color, and nucleocytoplasmic ratio in bladder cancer tissue and organoids are similar, suggesting that organoids and their source tissues are morphologically similar. Ki67 (a cell proliferation marker) immunofluorescence staining results ( Figure 3 The results showed that bladder cancer tissue contained Ki67-positive cells and had a high degree of tumor proliferation. The expression and distribution of Ki67 in bladder cancer organoids were similar to those in bladder cancer tissue.

[0070] The bladder cancer organoids used in this invention are not limited to the preparation method. The preparation method described in this basic embodiment is for illustrative purposes only. Bladder cancer organoids prepared by those skilled in the art based on this exemplary method should all fall within the protection scope of this invention.

[0071] Basic Example 2: Resuscitation of PBMCs from Healthy Individuals

[0072] Place the PBMC cryopreservation tubes in a 37°C water bath and gently shake until completely thawed (2 min). Add 18 mL of IMDM complete culture medium to the centrifuge tube, add PBMC, centrifuge at 500 g for 5 min, discard the supernatant, loosen the cell clumps by vibrating, add 20 mL of IMDM complete culture medium, and gently pipette 3-5 times to prepare a PBMC cell suspension.

[0073] Example 1 Cell Drugs

[0074] 1. Preparation of mixed culture medium: Bladder cancer culture medium and RPMI 1640 culture medium were mixed at a volume ratio of 1:1 to obtain mixed culture medium.

[0075] 2. Preparation of co-culture medium: IL-2, IL-12, anti-CD28 antibody and anti-PD-1 antibody were added to the mixed medium to obtain a co-culture medium containing 20 ng / mL IL-2, 5 ng / mL IL-12, 1 μg / mL anti-CD28 antibody and 10 μg / mL anti-PD-1 antibody.

[0076] 3. Preparation of organoid suspension: Collect bladder cancer organoids, centrifuge at 80g for 3 min, discard the supernatant, and resuspend in co-culture medium to 1×10⁻⁶. 4 The number of cells / mL was used to obtain an organoid suspension.

[0077] 4. Preparation of sensitized PBMC suspension: PBMCs were adjusted to a concentration of 1.5 × 10⁻⁶ using co-culture medium. 5 The concentration of cells / mL was increased to obtain a PBMC suspension. MUC1 peptide (final concentration of 5 μg / mL) was added, and the suspension was incubated at 37℃ and 5% CO2 for 24 h to obtain a sensitized PBMC suspension.

[0078] 5. Co-culture: Add organoid suspension (1×10⁻⁶) to each well of a preheated U-bottom 96-well plate. 4 Cells / mL), incubated at 37℃ in a 5% CO2 incubator for 2 hours. Sensitized PBMC suspension (1.5 × 10⁻⁶ cells / mL) was added according to a bladder cancer organoid: sensitized PBMC ratio of 1:15. 5 (cells / mL), gently pipet to mix, and co-culture at 37℃ and 5% CO2, denoted as D0.

[0079] 6. On days 5 and 10, gently aspirate the old culture medium with a pipette, add fresh co-culture medium, gently shake the plate once to distribute the medium evenly, and continue co-culturing; collect the cells on day 15, which are cytotoxic T cells (CTLs), referred to as cell drugs in this invention.

[0080] Example 2 Cellular Drugs

[0081] 1. Preparation of mixed culture medium: Bladder cancer culture medium and RPMI 1640 culture medium were mixed at a volume ratio of 1:1 to obtain mixed culture medium.

[0082] 2. Preparation of co-culture medium: IL-2, IL-12, anti-CD28 antibody and anti-PD-1 antibody were added to the mixed medium to obtain a co-culture medium containing 15 ng / mL IL-2, 10 ng / mL IL-12, 2 μg / mL anti-CD28 antibody and 15 μg / mL anti-PD-1 antibody.

[0083] 3. Preparation of organoid suspension: Collect bladder cancer organoids, centrifuge at 80g for 3 min, discard the supernatant, and resuspend in co-culture medium to 1×10⁻⁶. 4 The number of cells / mL was used to obtain an organoid suspension.

[0084] 4. Preparation of sensitized PBMC suspension: PBMC was adjusted to a concentration of 1.2 × 10⁻⁶ using co-culture medium. 5 The concentration of cells / mL was increased to obtain a PBMC suspension. MUC1 peptide (final concentration 6.5 μg / mL) was added and incubated at 37℃ in a 5% CO2 incubator for 24 h to obtain a sensitized PBMC suspension.

[0085] 5. Co-culture: Add organoid suspension (1×10⁻⁶) to each well of a preheated U-bottom 96-well plate. 4 Cells / mL), incubated at 37℃ in a 5% CO2 incubator for 2 hours. Sensitized PBMC suspension (1.2 × 10⁻⁶ cells / mL) was added according to a bladder cancer organoid: sensitized PBMC ratio of 1:12. 5 (cells / mL), gently pipet to mix, and co-culture at 37℃ and 5% CO2, denoted as D0.

[0086] 6. On days 5 and 10, gently aspirate the old culture medium with a pipette, add fresh co-culture medium, gently shake the plate once to distribute the medium evenly, and continue co-culturing; collect the cells on day 15, which are cytotoxic T cells (CTLs), referred to as cell drugs in this invention.

[0087] Example 3 Cellular Drugs

[0088] 1. Preparation of mixed culture medium: Bladder cancer culture medium and RPMI 1640 culture medium were mixed at a volume ratio of 1:1 to obtain mixed culture medium.

[0089] 2. Preparation of co-culture medium: IL-2, IL-12, anti-CD28 antibody and anti-PD-1 antibody were added to the mixed medium to obtain a co-culture medium containing 25 ng / mL IL-2, 7.5 ng / mL IL-12, 1.5 μg / mL anti-CD28 antibody and 7.5 μg / mL anti-PD-1 antibody.

[0090] 3. Preparation of organoid suspension: Collect bladder cancer organoids, centrifuge at 80g for 3 min, discard the supernatant, and resuspend in co-culture medium to 1×10⁻⁶. 4 The number of cells / mL was used to obtain an organoid suspension.

[0091] 4. Preparation of sensitized PBMC suspension: PBMCs were adjusted to a concentration of 1.8 × 10⁻⁶ using co-culture medium. 5 The cells / mL were used to obtain a PBMC suspension, and MUC1 peptide (final concentration 8 μg / mL) was added. The suspension was incubated at 37℃ and 5% CO2 for 24 h to obtain a sensitized PBMC suspension.

[0092] 5. Co-culture: Add organoid suspension (1×10⁻⁶) to each well of a preheated U-bottom 96-well plate. 4 Cells / mL), incubated at 37℃ in a 5% CO2 incubator for 2 hours. Sensitized PBMC suspension (1.8 × 10⁻⁶ cells / mL) was added according to a bladder cancer organoid: sensitized PBMC ratio of 1:18. 5 (cells / mL), gently pipet to mix, and co-culture at 37℃ and 5% CO2, denoted as D0.

[0093] 6. On days 5 and 10, gently aspirate the old culture medium with a pipette, add fresh co-culture medium, gently shake the plate once to distribute the medium evenly, and continue co-culturing; collect the cells on day 15, which are cytotoxic T cells (CTLs), referred to as cell drugs in this invention.

[0094] Comparative Example 1: Cellular Drugs

[0095] The only difference between Comparative Example 1 and Example 1 is step 2, "Preparation of Co-culture Medium". Step 2 of Comparative Example 1 is as follows:

[0096] 2. Preparation of co-culture medium: IL-15, IL-12, anti-CD28 antibody and anti-PD-1 antibody were added to the mixed medium to obtain a co-culture medium containing 20 ng / mL IL-15, 5 ng / mL IL-12, 1 μg / mL anti-CD28 antibody and 10 μg / mL anti-PD-1 antibody.

[0097] Comparative Example 2: Cellular Drugs

[0098] 1. Preparation of mixed culture medium: Bladder cancer culture medium and RPMI 1640 culture medium were mixed at a volume ratio of 1:1 to obtain mixed culture medium.

[0099] 2. Preparation of organoid suspension: Collect bladder cancer organoids, centrifuge at 80g for 3 min, discard the supernatant, and resuspend in mixed culture medium to a final volume of 1×10⁻⁶. 4 The number of cells / mL was used to obtain an organoid suspension.

[0100] 3. Adjust the concentration of PBMC to 1.5 × 10⁻⁶ using mixed culture medium. 5 The concentration of cells / mL was increased to obtain a PBMC suspension. MUC1 peptide (final concentration of 5 μg / mL) was added, and the suspension was incubated at 37℃ and 5% CO2 for 24 h to obtain a sensitized PBMC suspension.

[0101] 4. Co-culture: Add organoid suspension (1×10⁻⁶) to each well of a preheated U-bottom 96-well plate. 4 Cells / mL), incubated at 37℃ in a 5% CO2 incubator for 2 hours. Sensitized PBMC suspension (1.5 × 10⁻⁶ cells / mL) was added according to a bladder cancer organoid: sensitized PBMC ratio of 1:15. 5 (cells / mL), gently pipet to mix, and co-culture at 37℃ and 5% CO2, denoted as D0.

[0102] 5. On days 5 and 10, gently aspirate the old culture medium with a pipette, add fresh mixed culture medium, gently shake the plate once to distribute the culture medium evenly, and continue co-culturing; collect the cells on day 15, which are cytotoxic T cells (CTLs), referred to as cell drugs in this invention.

[0103] Comparative Example 3: Cell Drugs

[0104] The only difference between Comparative Example 3 and Example 1 is step 4, "Preparation of sensitized PBMC suspension". Step 4 of Comparative Example 3 is as follows:

[0105] 4. Preparation of sensitized PBMC suspension: PBMCs were adjusted to a concentration of 1.5 × 10⁻⁶ using co-culture medium. 5 The cells / mL were used to obtain a PBMC suspension, and MUC1 peptide (final concentration 15 μg / mL) was added. The suspension was incubated at 37℃ and 5% CO2 for 24 h to obtain a sensitized PBMC suspension.

[0106] Comparative Example 4: Cellular Drugs

[0107] The only difference between Comparative Example 4 and Example 1 is that steps 4-5 are different. Steps 4-5 of Comparative Example 4 are as follows:

[0108] 4. Preparation of sensitized PBMC suspension: PBMC was adjusted to a concentration of 2.0 × 10⁻⁶ using co-culture medium. 5 The concentration of cells / mL was increased to obtain a PBMC suspension. MUC1 peptide (final concentration of 5 μg / mL) was added, and the suspension was incubated at 37℃ and 5% CO2 for 24 h to obtain a sensitized PBMC suspension.

[0109] 5. Co-culture: Add organoid suspension (1×10⁻⁶) to each well of a preheated U-bottom 96-well plate. 4Cells / mL), incubated at 37℃ in a 5% CO2 incubator for 2 hours. Sensitized PBMC suspension (2.0 × 10⁻⁶ cells / mL) was added according to a bladder cancer organoid: sensitized PBMC ratio of 1:20. 5 (cells / mL), gently pipet to mix, and co-culture at 37℃ and 5% CO2, denoted as D0.

[0110] Comparative Example 5: Cell Drugs

[0111] 1. Preparation of mixed culture medium: Bladder cancer culture medium and RPMI 1640 culture medium were mixed at a volume ratio of 1:1 to obtain mixed culture medium.

[0112] 2. Preparation of organoid suspension: Collect bladder cancer organoids, centrifuge at 80g for 3 min, discard the supernatant, and resuspend in mixed culture medium to a final volume of 1×10⁻⁶. 4 The number of cells / mL was used to obtain an organoid suspension.

[0113] 3. Adjust the concentration of PBMC to 2.0 × 10⁻⁶ using mixed culture medium. 5 The cells / mL were used to obtain a PBMC suspension, and MUC1 peptide (final concentration 15 μg / mL) was added. The suspension was incubated at 37℃ and 5% CO2 for 24 h to obtain a sensitized PBMC suspension.

[0114] 4. Co-culture: Add organoid suspension (1×10⁻⁶) to each well of a preheated U-bottom 96-well plate. 4 Cells / mL), incubated at 37℃ in a 5% CO2 incubator for 2 hours. Sensitized PBMC suspension (2.0 × 10⁻⁶ cells / mL) was added according to a bladder cancer organoid: sensitized PBMC ratio of 1:15. 5 (cells / mL), gently pipet to mix, and co-culture at 37℃ and 5% CO2, denoted as D0.

[0115] 5. On days 5 and 10, gently aspirate the old culture medium with a pipette, add fresh mixed culture medium, gently shake the plate once to distribute the culture medium evenly, and continue co-culturing; collect the cells on day 15, which are cytotoxic T cells (CTLs), referred to as cell drugs in this invention.

[0116] Experimental Example 1: Cellular Drug Flow Cytometry Identification

[0117] In Examples 1-3 and Comparative Examples 1-5, the cells collected on day 15 were washed twice with PBS (containing 2% FBS), centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The cells were then resuspended in PBS to a concentration of 1×10⁻⁶. 6 Cells / mL, pass through a 70μm cell sieve, and add 100μL of cell suspension to a flow cytometer.

[0118] Add 100 μL of cell suspension to a flow cytometer, and add 5 μL of FITC-CD3 antibody, 5 μL of PE-CD8 antibody, and 5 μL of APC-CD107a antibody, respectively. Incubate at 4°C in the dark for 30 min.

[0119] Wash with 2 mL PBS (containing 2% FBS), centrifuge at 1500 rpm for 5 min, discard the supernatant; resuspend in 300 μL PBS (containing 2% FBS), and analyze using flow cytometry (BD FACS Canto II). Data are analyzed using FlowJo software. The results are shown in Table 1.

[0120] Table 1

[0121]

[0122] Experimental Example 2: The killing effect of cell-mediated drugs on bladder cancer organoids

[0123] In Examples 1-3 and Comparative Examples 1-5, cell-mediated drug (CTLs) collected on day 15 were used as effector cells in this experiment, and bladder cancer organoids from Basic Experiment 1 were used as target cells. Using a double staining method with CMTPX (red fluorescent labeling for bladder cancer organoids) and CFDA SE (green fluorescent labeling for CTLs), combined with the co-stimulatory effect of anti-CD28 coated culture plates, the killing process of CTLs on bladder cancer organoids (reduction in the number of bladder cancer organoids) and their self-activation and proliferation (increase in the number of CTLs) under an effector-target ratio of 5:1 were observed in real time, visually verifying the targeted killing ability of the cell-mediated drug.

[0124] 1. Dilute the anti-CD28 antibody to a final concentration of 5 μg / mL using antibody coating buffer. Add 100 μL of the diluted anti-CD28 antibody to each well of a 96-well black transparent plate and incubate at 4°C in the dark for 12 h. Discard the antibody solution in the wells, add 200 μL of PBS containing 2% BSA to each well, and block at room temperature for 1 h. Wash three times with PBS, allowing it to stand for 5 min each time, and discard the supernatant.

[0125] 2. Preparation of CMTPX-labeled bladder cancer organoid suspension: Centrifuge 80g of bladder cancer organoids for 3 min, discard the supernatant, gently resuspend twice in PBS, and dilute the CMTPX dye to a final concentration of 5 μM with co-culture medium. Add CMTPX dye at a concentration of 1 × 10⁻⁶ μL per 10⁻⁶ μL. 4 100 μL of dye solution was added to each bladder cancer organoid, and the mixture was incubated at 37°C with 5% CO2 for 30 min. Incubation was terminated by adding 1 mL of co-culture medium, and the mixture was centrifuged at 80 g for 3 min, discarding the supernatant. The bladder cancer organoids were resuspended in co-culture medium to a concentration of 2 × 10⁻⁶. 3 cells / mL (calculated based on an efficacy-to-target ratio of 5:1, subsequent wells require 2 × 10⁶ bladder cancer organoids).3 One, corresponding to CTL 1×10 4 indivual);

[0126] 3. Preparation of CFDASE-labeled CTL suspension: The D15 cell drugs from Examples 1-3 or Comparative Examples 1-5 were centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the cells were washed twice with PBS and resuspended to a concentration of 1×10⁻⁶. 6 The concentration of CTLs was increased to 1000 cells / mL to obtain a preliminary CTL suspension. CFDASE dye was diluted to a final concentration of 5 μM with co-culture medium and mixed with the CTL suspension at a 1:1 volume ratio. The mixture was incubated at 37°C with 5% CO2 for 20 min. Five volumes of co-culture medium (containing 10% FBS) were added, and the mixture was allowed to stand at room temperature for 10 min. The mixture was centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The CTLs were resuspended in co-culture medium to a concentration of 1 × 10⁻⁶ cells / mL. 4 cells / mL.

[0127] 4. Add 100 μL of CMTPX-labeled bladder cancer organoid suspension and 100 μL of CFDASE-labeled CTL suspension of Examples 1-3 or Comparative Examples 1-5 to each well of an anti-CD28-coated 96-well plate. At the same time, set up an organoid control group (100 μL of CMTPX-labeled bladder cancer organoid suspension + 100 μL of co-culture medium) as a control. Incubate at 37°C and 5% CO2 for 72 h.

[0128] 5. Observe the degree of quenching of red fluorescent markers (killing effect) in bladder cancer organoids and the penetration of CTL green fluorescence into the red area. Use ImageJ software to analyze the imaging images at each time point, select 3 random fields of view, and measure the integrated optical density (IOD) of red fluorescence in bladder cancer organoids and the IOD of green fluorescence in CTL.

[0129] Organoid survival index = (72h red IOD of the kill group / 0h red IOD of the kill group) × 100%;

[0130] CTL proliferation index = (72h green IOD of the kill group / 0h green IOD of the kill group) × 100%;

[0131] The lower the organoid survival index, the stronger the killing effect; the higher the CTL proliferation index, the more obvious the activation and proliferation.

[0132] The measurement results are shown in Table 2:

[0133] Table 2

[0134]

[0135] Experimental Example 3: Evaluation of the efficacy of cell-based drugs in anticancer drugs

[0136] 1. Dilute the anti-CD28 antibody to a final concentration of 5 μg / mL using antibody coating buffer. Add 100 μL of the diluted anti-CD28 antibody to each well of a 96-well black transparent plate and incubate at 4°C in the dark for 12 h. Discard the antibody solution in the wells, add 200 μL of PBS containing 2% BSA to each well, and block at room temperature for 1 h. Wash three times with PBS, allowing it to stand for 5 min each time, and discard the supernatant.

[0137] 2. Preparation of CMTPX-labeled bladder cancer organoid suspension: Refer to Experimental Example 2;

[0138] 3. Preparation of CFDASE-labeled CTL suspension: The D15 cell drug from Example 1 was centrifuged at 1500 rpm for 5 min, the supernatant was discarded, the cells were washed twice with PBS, and resuspended to a concentration of 1×10⁻⁶. 6 The concentration of CTLs was increased to 1000 cells / mL to obtain a preliminary CTL suspension. CFDASE dye was diluted to a final concentration of 5 μM with co-culture medium and mixed with the CTL suspension at a 1:1 volume ratio. The mixture was incubated at 37°C with 5% CO2 for 20 min. Five volumes of co-culture medium (containing 10% FBS) were added, and the mixture was allowed to stand at room temperature for 10 min. The mixture was centrifuged at 1500 rpm for 5 min, and the supernatant was discarded. The CTLs were resuspended in co-culture medium to a concentration of 1 × 10⁻⁶ cells / mL. 4 cells / mL.

[0139] 4. Drug gradient setting: The candidate drug (erdatinib) was diluted to three concentration gradients (10, 100, 1000 nM) in co-culture medium, and an organoid control group and a CTL control group were set up at the same time.

[0140] 5. Vaccination system:

[0141] Low drug concentration group: 100 μL CMTPX-labeled organoid suspension + 50 μL 10 nM drug + 50 μL CFDASE-labeled CTL suspension.

[0142] Drug concentration group: 100 μL CMTPX-labeled organoid suspension + 50 μL 100 nM drug + 50 μL CFDASE-labeled CTL suspension.

[0143] High drug concentration group: 100 μL CMTPX-labeled organoid suspension + 50 μL 1000 nM drug + 50 μL CFDASE-labeled CTL suspension.

[0144] Organoid control group: 100 μL CMTPX-labeled organoid suspension + 100 μL co-culture medium;

[0145] CTL control group: 100 μL CFDA SE-labeled CTL suspension + 100 μL co-culture medium;

[0146] Incubate at 37℃ in a 5% CO2 incubator for 72 hours.

[0147] 6. Observe the degree of quenching of red fluorescent markers (killing effect) in bladder cancer organoids and the penetration of CTL green fluorescence into the red area. Use ImageJ software to analyze the imaging images at each time point, select 3 random fields of view, and measure the integrated optical density (IOD) of red fluorescence in bladder cancer organoids and the IOD of green fluorescence in CTL.

[0148] Organoid survival index = (Red IOD in the drug group at 72h / Red IOD in the drug group at 0h) × 100%

[0149] CTL proliferation index = (72h green IOD in drug group / 0h green IOD in drug group) × 100%

[0150] The lower the organoid survival index, the stronger the killing effect; the higher the CTL proliferation index, the more obvious the activation and proliferation.

[0151] The measurement results are shown in Table 3:

[0152] Table 3

[0153]

[0154] As shown in Table 3, the organoid survival index decreased in a dose-dependent manner with increasing drug concentration, and the high-concentration group (1000 nM) was 62.15% lower than that of the Example 1 group in Experiment 2 (28.35±2.76); the CTL proliferation index increased in a dose-dependent manner with increasing drug concentration, and the high-concentration group was 51.84% higher than that of the Example 1 group in Experiment 2 (363.83±20.57).

[0155] The above results demonstrate that the bladder cancer drug efficacy evaluation model (CTL-bladder cancer organoid co-culture model) of this invention can simultaneously capture the direct antitumor and immunomodulatory effects of drugs, overcoming the limitation of traditional models in evaluating the "tumor microenvironment-immune cell interaction." The evaluation results of the bladder cancer drug efficacy evaluation model provided by this invention can be directly correlated with clinical efficacy, providing key predictive evidence for whether a drug is worth pursuing in clinical research, and avoiding the risk of conversion from basic efficacy to clinical ineffectiveness.

[0156] The above detailed description is a specific illustration of one feasible embodiment of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or modifications made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A cell-based drug, characterized in that, The method for preparing the cell-based drug includes the following steps: S1. Bladder cancer organoids were resuspended in co-culture medium to obtain organoid suspensions; S2 and PBMC were resuspended in co-culture medium to obtain PBMC suspension. MUC1 peptide was added to make the final concentration of MUC1 peptide in PBMC suspension 5-8 μg / mL. After incubation, sensitized PBMC suspension was obtained. S3. According to the cell ratio of bladder cancer organoids to sensitized PBMCs = 1:12-18, add sensitized PBMC suspension to organoid suspension, co-culture, collect cells after co-culture, and obtain cell drug; The co-culture medium contains 15-25 ng / mL IL-2, 5-10 ng / mL IL-12, 1-2 μg / mL anti-CD28 antibody and 7.5-15 μg / mL anti-PD-1 antibody; The co-culture medium is prepared by mixing organoid culture medium and immunobasal medium, and then adding IL-2, IL-12, anti-CD28 antibody and anti-PD-1 antibody to obtain co-culture medium; the organoid culture medium is used for the culture of bladder cancer organoids; the immunobasal medium is used for the culture of PBMCs; the volume ratio of organoid culture medium to immunobasal medium is 1:0.1-10. The organoid culture medium is a medium containing 25-75 ng / mL recombinant human epidermal growth factor, 75-125 ng / mL recombinant human Noggin, 800-1200 ng / mL recombinant human R-spondin 1, 400-600 ng / mL recombinant human FGF 10, 15-25 ng / mL recombinant human FGF-basic, and 3-7 μM A83-01.

2. The cell-based drug according to claim 1, characterized in that, The immune basal culture medium includes any one or more of RPMI 1640 medium, IMDM medium, DMEM medium, DMEM / F12 medium, MEM medium, α-MEM medium, and Ham's F-12 medium.

3. The cell-based drug according to claim 1, characterized in that, The incubation in step S2 includes incubation at 35-40℃ for 24-48 hours; the co-cultivation in step S3 includes co-cultivation at 35-40℃ for 15-30 days.

4. The method for preparing the cell drug according to any one of claims 1-3.

5. The application of the cell-based drug according to any one of claims 1-3 in the preparation of a bladder cancer pharmacodynamic evaluation model, characterized in that, The method for preparing the bladder cancer drug efficacy evaluation model is as follows: co-culture cellular drugs with bladder cancer organoids to obtain the bladder cancer drug efficacy evaluation model.

6. The bladder cancer efficacy evaluation model obtained from the preparation of cell drugs according to any one of claims 1-3.

7. A method for evaluating the efficacy of a bladder cancer drug, characterized in that, The efficacy evaluation method includes using the cell drug as described in any one of claims 1-3 or the bladder cancer efficacy evaluation model as described in claim 6.

8. The efficacy evaluation method according to claim 7, characterized in that, The efficacy evaluation methods mentioned above include: (1) A bladder cancer drug efficacy evaluation model was obtained by co-culturing cell drugs with bladder cancer organoids; (2) Use a bladder cancer drug treatment efficacy evaluation model to evaluate the anti-bladder cancer efficacy of bladder cancer drugs.

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