Method for constructing RC48 acquired drug resistance in-situ gastric cancer mouse model and RC48 acquired drug resistance in-situ gastric cancer mouse model

Abstract

The invention relates to the field of tumor animal models, in particular to a method for constructing an RC48 acquired drug-resistant in-situ gastric cancer mouse model and the RC48 acquired drug-resistant in-situ gastric cancer mouse model. The method comprises the following steps: S1, transplanting a tumor organ to the stomach of a target animal, carrying out animal living body imaging, and after a living body imaging numerical value reaches a certain threshold value, carrying out drug administration by using RC48; s2, performing animal living body imaging, drawing a tumor growth curve, killing the animal when the tumor growth curve is changed from a descending trend to a rising trend and an imaging numerical value after subsequent medication is not reduced any more, and performing digestion culture on the tumor tissue again to form a drug-resistant tumor organ; s3, transplanting the drug-resistant tumor organoid, so as to obtain the RC48 acquired drug-resistant in-situ gastric cancer mouse model. The animal model method constructs a drug-resistant animal model with complete immunization, in-situ tumor and RC48 drug treatment, is closer to the actual situation of a real case, and has more excellent guiding significance for drug research and development.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering technology, and in particular to a method for constructing an RC48 acquired drug-resistant orthotopic gastric cancer mouse model and the constructed animal model, belonging to the field of tumor animal models. Background Technology

[0002] Existing studies have all involved treating cell lines with HER2-ADC drugs for extended periods to achieve acquired drug resistance, followed by subcutaneous transplantation into mice to explore in vitro and in vivo mechanisms. RC48, a HER2-ADC drug developed by Rongchang Pharmaceuticals, is currently recommended as a first-line treatment for HER2-positive gastric cancer patients due to its price and clinical trial efficacy.

[0003] Antitumor drugs often lose their therapeutic effect due to the rapid development of drug resistance. Accurately assessing the development and changes in drug resistance is crucial for standardized clinical medication use and maximizing the therapeutic effect on patients, thus improving patient survival. Currently, there are no reports on RC48 resistance research, and the technology for constructing animal models of RC48 resistance is also lacking.

[0004] The following patents and articles were found regarding existing technologies for animal models of gastric cancer.

[0005] Related patents: 1) Chinese invention patent CN114480250B, "Method for Constructing an Orthotopic Primary Gastric Cancer Animal Model", ZL 2020 11266740.1, constructs an orthotopic primary gastric cancer model, but this patent does not involve drug resistance research in mouse gastric cancer models and does not achieve complete immune capacity in mice. Therefore, it cannot be used for RC48 resistance research at this time.

[0006] 2) Chinese invention patent application CN119564369A, "A method for establishing and applying a mouse gastric cancer orthotopic transplantation model", application number CN202411759589.3. This patent also constructs a mouse gastric cancer model, but this patent involves the injection of gastric adenocarcinoma cell lines. The construction speed of the mouse gastric cancer model is relatively slow and requires a long feeding time.

[0007] 3) Chinese invention patent application CN 120210124 A, "A Human Bladder Cancer Vidicetuximab-Resistant Cell Line and Its Construction Method and Application," addresses the issue that acquired resistance to ADC drugs is almost always encountered in the treatment of most tumors. To assess the drug resistance of bladder cancer cells, human bladder transitional cell carcinoma T24 cells were used as the induction target. A method of low-concentration continuous induction and gradual increase in drug concentration and treatment time was employed, inducing and culturing cells for 5 months to construct a human bladder transitional cell carcinoma cell line resistant to RC48. This patented method targets bladder cancer cells and uses an in vitro culture method. It has limitations due to the long induction and culture period of T24 cells, and the bladder cancer cell line is not in situ carcinoma. Therefore, when constructing animal models for colonization, there is a risk of unstable colonization rates and difficulty in controlling the consistency of the animal model.

[0008] Related articles: 1) PMID:38177458: A mouse model of gastric cancer was constructed.

[0009] 2) PMID:37620320: The human HER2 gene was overexpressed in the cell line and transplanted subcutaneously into mice. The transplanted mice were treated with RC48, but the treatment did not reach the drug resistance stage. Summary of the Invention

[0010] The purpose of this invention is to overcome the lack of drug-resistant gastric cancer models in the existing technology, and to provide a method and model for constructing an RC48 acquired drug-resistant in situ gastric cancer animal model.

[0011] In a first aspect, the present invention provides a method for constructing an animal model.

[0012] A method for constructing an animal model of RC48-acquired drug-resistant orthotopic gastric cancer includes: S1. Transplant tumor organoids into the stomach of the target animal, perform in vivo imaging of the animal, and administer RC48 when the in vivo imaging values ​​reach a certain threshold. S2. Perform in vivo imaging of the animal and plot the tumor growth curve. When the tumor growth curve changes from a downward trend to an upward trend, and the imaging values ​​no longer decrease after subsequent drug administration, the small animal can be euthanized, and the tumor tissue can be digested and cultured again to produce drug-resistant tumor organoids.

[0013] The time interval for in vivo animal imaging is shorter than the time interval for tail vein injection.

[0014] S3. Transplanting drug-resistant tumor organoids yields the RC48 acquired drug-resistant orthotopic gastric cancer mouse model.

[0015] This invention transplants gastric tumor organoids into the stomach of small animals, allowing them to colonize and form tumors, simulating the in situ immune microenvironment. After continuous in vivo administration of RC48, an RC48-acquired drug resistance animal model is obtained, simulating the entire process of gastric cancer patients from RC48 sensitivity to drug resistance. This method shortens the processing time and provides a complete immune microenvironment, making it a powerful research tool.

[0016] Furthermore, in S1, the target animal is a C57 mouse. Tumor organoids are transplanted into the stomach of the target animal, and in vivo imaging is performed 1-5 days later. When the value reaches 3×10⁻⁶... 6 -2×10 7 When administering the medication, use RC48 at a dose of 1.6-5 mg / kg per animal, administered via tail vein injection every 3-7 days.

[0017] Furthermore, in S1, after the tumor is transplanted into the stomach of the target animal, in vivo imaging of the animal is performed three days later.

[0018] Furthermore, in S1, the dosage is 2.5-4.5 mg / kg per animal.

[0019] Furthermore, in S1, the drug is administered via tail vein injection every four to five days.

[0020] Furthermore, in S2, live animal imaging is performed every three to five days.

[0021] Furthermore, in S2, the change from a downward trend to an upward trend means that after RC48 administration, the measured trend of tumor change changes from decreasing to increasing. This requires first observing a decrease in in vivo imaging values, and then observing an increase in imaging values.

[0022] If subsequent drug administration results in no further decrease in imaging values, it indicates acquired drug resistance in gastric cancer tissue resulting from in situ growth of organoids. Alternatively, treatment can be stopped once the tumor imaging values ​​cease to change. Since in vivo imaging indirectly characterizes tumor tissue and cannot obtain the actual tumor tissue, resistance is considered acquired upon confirmation that tumor imaging values ​​no longer decrease.

[0023] Furthermore, in S2, an upward trend in the tumor growth curve indicates that the tumor tissue begins to increase significantly, and the significant increase is defined as an increase rate of ≥30% in two consecutive animal in vivo imaging values.

[0024] Furthermore, in S2, when the imaging values ​​no longer decrease after subsequent drug administration, the animal can be euthanized, and the tumor tissue can be digested and cultured again to produce drug-resistant tumor organoids.

[0025] Furthermore, the gastric tissue is Trp53 - / - Normal gastric tissue of Cas9-EGFP animals.

[0026] Preferably, the gastric tissue is human gastric tissue.

[0027] Furthermore, in S1, the tumor organoids are obtained by the following method: Stomach tissue was obtained, digested, and cultured into organoids. KRAS G12D amplification and Pten gRNA mutation were introduced, and the tissue was transplanted into the stomach of a C57-immunized animal. After tumor formation, the tumor tissue was removed, digested with collagenase, and cultured into tumor organoids.

[0028] In a second aspect, the present invention provides an animal model of RC48 acquired drug-resistant in situ gastric cancer.

[0029] The RC48 acquired drug-resistant in situ gastric cancer animal model was obtained using the above method.

[0030] Preferably, the animal model is a mouse model. More preferably, the mouse model is a C57 mouse model.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. The animal model method of this invention constructs an immune-complete, in situ tumor-resistant animal model treated with RC48 drug, which is closer to the actual situation of real cases and has better guiding significance for drug development.

[0032] 2. This invention provides an RC48-acquired drug resistance mouse model that simulates the entire process of gastric cancer patients from RC48 sensitivity to drug resistance. This method shortens the treatment time and has a complete immune microenvironment, making it a powerful research tool. Attached Figure Description

[0033] Figure 1 This is a flowchart illustrating Embodiment 1 of the present invention.

[0034] Figure 2 This is a growth curve diagram after drug administration to a subcutaneous tumor transplant.

[0035] Figure 3 Images of tumor organoids from the control and experimental groups.

[0036] Figure 4 This is a tumor growth curve for in-situ drug administration. Detailed Implementation

[0037] The present invention uses C57 mice for detailed experimental method description, but is not limited to using C57 mice as the basis of animal model. Those skilled in the art may also use other equivalent or similar animals as the basis of model.

[0038] C57 mice, also known as C57BL / 6, are a common inbred strain of laboratory mice. They are widely used in genetic experiments as transgenic mice to simulate genetic defects in humans. Due to their versatility as a single strain, ease of breeding, and robust physique, they are widely applicable, have high sales volume, and are a breed that readily provides stable and reliable laboratory animals.

[0039] HER2 is an abbreviation for Human Epidermal Growth Factor Receptor 2, and ADC is an abbreviation for Antibody-Drug Conjugate. HER2-ADC drugs specifically refer to antibody-drug conjugates that target the HER2 receptor.

[0040] RC48, or Vidicetumab, is my country's first independently developed innovative antibody-drug conjugate developed by Rongchang Biopharmaceutical (Yantai) Co., Ltd. Vidicetumab for injection (trade name: Aidixi).

[0041] Existing studies have all involved treating cell lines with HER2-ADC drugs and then subjecting them to long-term drug treatment to achieve drug-acquired resistance. These cells were then transplanted subcutaneously into mice to explore in vitro and in vivo mechanisms. RC48, a HER2-ADC drug developed by Rongchang Company, is currently recommended as a first-line clinical treatment for HER2-positive gastric cancer patients due to its price and clinical trial results.

[0042] The present invention transplants gastric tumor organoids into the stomach of mice, allowing them to colonize and form tumors, simulating the in situ immune microenvironment. After continuous in vivo administration of RC48, an RC48-acquired resistance mouse model is finally created, simulating the entire process of gastric cancer patients from RC48 sensitivity to drug resistance. This method shortens the treatment time and provides a complete immune microenvironment, making it a powerful research tool.

[0043] Specifically, mouse animal models can be constructed according to the following time parameters.

[0044] 1) Short treatment time: RC48 drug treatment is performed three days after transplantation, and the drug is administered once every five days. Within one month, an RC48-acquired drug-resistant mouse gastric cancer model can be obtained, which goes through the entire process from sensitivity to drug resistance.

[0045] 2) It has a complete immune microenvironment: When tumor organoids are transplanted into the stomach of mice, this site is closer to the immune microenvironment system of gastric cancer patients than in vitro and subcutaneously.

[0046] 3) RC48, as a novel domestic HER2-ADC drug, has been included in clinical drug use guidelines and is cheaper than the foreign-made DS-8201 (trastuzumab, Roche), with a wider range of application prospects. Therefore, the emergence of acquired drug resistance will be more rapid and widespread, and a research tool is urgently needed to analyze the relevant mechanisms. Previous research tools and studies have focused on the tumor cells themselves, but we all know that the process of drug resistance is a process that affects cells, and a fully immune in vivo environment is needed to better characterize patient data.

[0047] In addition to exploring drug resistance mechanisms, this invention can also be used to verify possible combination therapy strategies to reverse drug resistance, as well as biological markers of acquired resistance to RC48 drugs.

[0048] The present invention will now be described in further detail with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0049] Example 1 A model was constructed using humanized HER2 mouse gastric tissue.

[0050] like Figure 1 As shown, humanized HER2 mice were first combined with Trp53 - / - Humanized HER2 homozygotes were obtained by hybridization of Cas9-EGFP mice; Trp53 - / - Normal gastric tissue was obtained from Cas9-EGFP mice, digested, and cultured into organoids. KRAS G12D amplification and Pten gRNA mutation were then introduced, and the tissue was transplanted into the stomachs of C57-immunized mice. After tumor formation, tumor tissue was removed and digested with collagenase on a shaker at 37°C. The digestion process was terminated when a clear single-cell state was observed under a microscope. After centrifugation, the tissue was mixed with matrix gel and a dedicated organoid culture medium (prepared according to the organoid culture medium components disclosed in Example 1 of Chinese Invention Patent CN114480250B) was added to culture tumor organoids.

[0051] Tumor organoids were transplanted into the stomachs of C57 mice, and in vivo imaging of the small animals was performed three days later. When the value reached 10... 6 The RC48 drug was prepared as a 0.6 mg / mL solution using physiological saline. The RC48 dose was 2.5 mg / kg body weight, administered via tail vein injection to each experimental mouse every five days. In vivo imaging was performed every three days to plot tumor growth curves. Figure 2As shown in the figure, the growth curve after subcutaneous tumor transplantation shows that RC48 showed a significant inhibitory effect in the early stage, but resistance was subsequently developed and the tumor tissue grew rapidly.

[0052] When the tumor growth curve changes from a downward trend to an upward trend, and the imaging data increases significantly, the mouse can be euthanized, and the tumor tissue can be digested and cultured again to produce drug-resistant tumor organoids for transplantation. After in vivo imaging detection of the animal as above, the drug is administered, and the tumor growth curve is plotted. If the curve does not decrease but continues to rise, it proves that the acquired drug resistance model has been successfully constructed.

[0053] like Figure 3 As shown in the images, tumor organoids in the control and experimental groups are grown. In the micrographs, the cells in the experimental group have more diverse morphologies and are more sparse. The drug significantly inhibits cell proliferation and induces changes in cell morphology, indicating that RC48 has a killing effect on tumor cells in the experimental group, and the surviving cells are drug-resistant tumor cells.

[0054] like Figure 4 As shown in the figure, the tumor growth curve after in situ administration shows that the tumor imaging values ​​first decreased and then increased after administration, indicating that the tumor tissue in the experimental group acquired drug resistance.

[0055] Thus, the RC48 drug-resistant mouse model was obtained.

[0056] Example 2 A mouse model was constructed following the method described in Example 1, with the only difference being that the dose administered via the tail vein was 1.5 mg / kg. The tumors did not shrink and grew as rapidly as in the untreated group.

[0057] Example 3 The drug-resistant mouse model obtained in Example 1 by transplanting drug-resistant tumor organoids was cultured for 30 days, and the volume of the tumor tissue was detected by in vivo imaging. The average value was 1x10⁻⁶. 7 p / sec / cm 2 / sr indicates that the volume of drug-resistant tumors in all mouse models that underwent transplantation of drug-resistant tumor organoids was consistent, and all mice were stable and reliable for drug development research.

[0058] Example 4 like Figure 1 As shown, obtain Trp53 - / -Normal gastric tissue from Cas9-EGFP mice was digested, cultured into organoids, and then inoculated with KRAS G12D amplification and Pten gRNA mutation before being transplanted into the stomachs of C57-immunized mice. After tumor formation, tumor tissue was removed and digested with collagenase on a 37-degree shaker. The digestion process was terminated when a clear single-cell state was observed under a microscope. After centrifugation, the tissue was mixed with matrix gel and cultured in a dedicated organoid culture medium to produce tumor organoids.

[0059] Tumor organoids were transplanted into the stomach of C57 animals. Three days later, in vivo imaging of the small animals was performed. When the value reached 10 to the power of 6, the drug was administered. RC48 was prepared into a solution of 0.6 mg / mL with physiological saline. The drug was administered via tail vein injection at a dose of 2.5 mg / kg per animal, once every five days. In vivo imaging of the small animals was performed every three days to plot the tumor growth curve.

[0060] Because the cells are not humanized HER2 cells, there are no drug targets, and this tumor model cannot respond to drugs. Tumor tissues in both the control and experimental groups grew significantly, making it impossible to construct a drug-resistant animal model.

[0061] Comparative Example 1 Using Chinese invention patent CN114480250B, "Method for constructing an in situ primary gastric cancer animal model", an in situ primary gastric cancer model was constructed. The tumor cells of the resulting mouse gastric cancer model did not have RC48 resistance and could not be used for RC48 resistance research and development.

[0062] Comparative Example 2 The mouse gastric cancer model constructed using Chinese invention patent application CN119564369A, entitled "A Method for Establishing and Applying a Mouse Orthotopic Transplantation Model of Gastric Cancer," requires in vitro gene editing to obtain a gastric adenocarcinoma cell line. This process involves a lengthy culture time for the gastric adenocarcinoma cells, necessitating multiple matings and passages in mice. The resulting cells are not orthotopic cells from the mouse stomach, and the gastric adenocarcinoma is not in situ. After injection of the obtained gastric adenocarcinoma cell line into the mouse stomach, it needs to be cultured continuously for 25-30 days to obtain the mouse gastric cancer model.

[0063] Although the injected gastric adenocarcinoma cell lines were selected drug-resistant cell lines, these cell lines were not gastric cancer cells that occurred in situ. After being injected into the stomach of mice, they needed to be cultured continuously for a long time before they could colonize. Furthermore, when culturing mouse models in large quantities, individual differences were likely to occur, and some mice could not be transformed into gastric cancer models. The volume of gastric adenocarcinoma varied greatly among different mice after being cultured for the same period of time.

[0064] 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, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0065] Furthermore, the use of terms such as "first," "second," and "third" in terminology is merely for distinguishing descriptions of identical or similar components and should not be interpreted as emphasizing or implying the relative importance of a particular component.

[0066] Furthermore, in the description of the embodiments of the present invention, "several", "more than", and "a number of" represent at least two. The number can be any number, such as two, three, four, five, six, seven, eight, or nine, and can even exceed nine.

Claims

1. A method for constructing an animal model of RC48-acquired drug-resistant orthotopic gastric cancer, characterized in that, include: S1. Transplant tumor organoids into the stomach of the target animal, perform in vivo imaging of the animal, and administer RC48 when the in vivo imaging values ​​reach a certain threshold. S2. Perform in vivo imaging of animals and plot tumor growth curves. When the tumor growth curve changes from a downward trend to an upward trend, and the imaging values ​​no longer decrease after subsequent drug administration, the small animals can be euthanized, and the tumor tissue can be digested and cultured again to produce drug-resistant tumor organoids. The time interval for in vivo animal imaging is shorter than the time interval for tail vein injection; S3. Transplanting drug-resistant tumor organoids yields the RC48 acquired drug-resistant orthotopic gastric cancer mouse model.

2. The method for constructing an RC48-acquired drug-resistant orthotopic gastric cancer animal model according to claim 1, characterized in that, In S1, the target animal is a C57 mouse; tumor organoids are transplanted into the stomach of the target animal, and in vivo imaging is performed 1-5 days later. When the value reaches 3×10 6 -2×10 7 When administering the medication, use RC48 at a dose of 1.6-5 mg / kg per animal, administered via tail vein injection every 3-7 days.

3. The method for constructing an RC48-acquired drug-resistant orthotopic gastric cancer animal model according to claim 1, characterized in that, In S1, after the tumor is transplanted into the stomach of the target animal, in vivo imaging of the animal is performed three days later.

4. The method for constructing an RC48-acquired drug-resistant orthotopic gastric cancer animal model according to claim 1, characterized in that, In S1, the drug is administered via tail vein injection every four to five days.

5. The method for constructing an RC48-acquired drug-resistant orthotopic gastric cancer animal model according to claim 1, characterized in that, In S2, live animal imaging is performed every three to five days.

6. The method for constructing an RC48-acquired drug-resistant orthotopic gastric cancer animal model according to claim 1, characterized in that, In S2, the change from a downward trend to an upward trend means that after RC48 administration, the measured trend of tumor change changed from decreasing to increasing.

7. The method for constructing an RC48-acquired drug-resistant orthotopic gastric cancer animal model according to claim 1, characterized in that, In S2, an upward trend in the tumor growth curve indicates that the tumor tissue begins to increase significantly, and the significant increase is defined as an increase rate of ≥30% in two consecutive animal in vivo imaging values.

8. The method for constructing an RC48-acquired drug-resistant orthotopic gastric cancer animal model according to claim 1, characterized in that, The stomach tissue is Trp53 - / - Normal gastric tissue of Cas9-EGFP animals.

9. The method for constructing an animal model of RC48 acquired drug-resistant orthotopic gastric cancer according to claim 1, characterized in that, In S1, the tumor organoids are obtained by the following method: obtaining gastric tissue, digesting it and culturing it into organoids; introducing KRAS G12D amplification and Pten gRNA mutation, transplanting it into the stomach of a C57-immunized animal, and after tumor formation, removing the tumor tissue and digesting it with collagenase to culture it into tumor organoids.

10. An animal model of RC48 acquired drug-resistant in situ gastric cancer, obtained by the method described in any one of claims 1-9.

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