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CN-121970726-A - PD-1/PD-L1 immune treatment acquired drug-resistant mouse subcutaneous tumor model construction method based on in-vivo multidimensional immune landscape evolution

CN121970726ACN 121970726 ACN121970726 ACN 121970726ACN-121970726-A

Abstract

The invention discloses a method for constructing a PD-1/PD-L1 immune therapy acquired drug-resistant mouse subcutaneous tumor model based on in-vivo multidimensional immune landscape evolution, and relates to the field of animal model construction; the method comprises the following steps of preparing tumor cells, recovering and amplifying MC38 colon adenocarcinoma cell strains, preparing single cell suspensions, establishing a basic model, inoculating the MC38 single cell suspensions under the skin of a syngeneic C57BL/6 mouse, establishing a tumor-bearing mouse model, and performing drug resistance induction and screening, namely applying continuous treatment pressure of an anti-PD-1 or anti-PD-L1 antibody to the tumor-bearing mouse after the tumor grows to a preset volume. First, the tumor volume of the mice in the treatment group is compared with the average volume of the control group at the same time point, and the response mice with the tumor volume remarkably smaller than that of the control group are screened out, so that primary drug resistant individuals are excluded. Dynamic immunity monitoring sampling, sample processing and analysis. The invention has the characteristics of repeatability and dynamic monitoring, and overcomes the defects of the existing preclinical model.

Inventors

  • YANG KE
  • HAO JIANGTAO

Assignees

  • 重庆医科大学

Dates

Publication Date
20260505
Application Date
20260210

Claims (8)

  1. 1. The method for constructing the PD-1/PD-L1 immune treatment acquired drug-resistant mouse subcutaneous tumor model based on in-vivo multidimensional immune landscape evolution is characterized by comprising the following steps of: s1, preparing tumor cells, namely resuscitating and amplifying MC38 colon adenocarcinoma cell lines to prepare single-cell suspension; S2, establishing a basic model, namely inoculating MC38 single cell suspension to the subcutaneous of a homologous C57BL/6 mouse, and establishing a tumor-bearing mouse model; S3, drug resistance induction and screening, namely, after the tumor grows to a preset volume, applying continuous treatment pressure of an anti-PD-1 or anti-PD-L1 antibody to the tumor-bearing mice, firstly, comparing the tumor volume of the mice in the treatment group with the average volume of a control group at the same time point, screening out a 'response mouse' with the tumor volume obviously smaller than that of the control group, and eliminating primary drug resistant individuals; S4, dynamic immunity monitoring sampling, namely, in the treatment process of the step S3, non-lethal peripheral blood collection and tumor biopsy sampling are carried out on the same mouse individual according to a preset time point; s5, sample treatment and analysis, namely preparing the sample obtained in the step S4 into single-cell suspension, analyzing the immune microenvironment characteristics through flow cytometry, and verifying the drug resistance phenotype.
  2. 2. The method for constructing the subcutaneous tumor model of the PD-1/PD-L1 immune treatment acquired drug-resistant mice based on the in-vivo multidimensional immune landscape evolution according to claim 1, wherein in the step S3, the specific scheme of drug resistance induction is that the drug administration is started when the volume of the subcutaneous tumor reaches 50-100mm3, the dosage and the frequency of the drug administration are that the anti-PD-1 antibody or the anti-PD-L1 antibody is injected intraperitoneally, the dosage is 5-10mg/kg or 200 mug/dose, the frequency is once every 3 days, the drug administration is continued until the drug resistance judgment end point is reached, and the ethical end point is judged when the volume of the tumor reaches 2000mm 3.
  3. 3. The method for constructing a subcutaneous tumor model of a PD-1/PD-L1 immune treatment-acquired drug-resistant mouse based on in vivo multidimensional immune landscape evolution according to claim 1, wherein in the step S3, the decision criteria of the acquired drug-resistant model are simultaneously the following conditions, namely initial response confirmation that the tumor volume of the mouse regresses or the disease is stable and the volume is significantly lower than that of a control group in the early stage of treatment, drug-resistant progress confirmation that the tumor volume is continuously measured and increased 3 times under the continuous administration condition, a volume threshold that the tumor volume rebounds to 5 times higher than the initial volume, and tumor volume monitoring is measured by using a vernier caliper, wherein the calculation formula is that the tumor volume=1/2 square of length×width.
  4. 4. The method for constructing the subcutaneous tumor model of the PD-1/PD-L1 immune therapy acquired drug resistant mice based on in-vivo multidimensional immune landscape evolution according to claim 1, wherein in the step S4, 5-20mg of tumor core tissues are obtained by biopsy needle puncture or minimally invasive surgery on the premise of not killing the mice.
  5. 5. The method for constructing a subcutaneous tumor model of a PD-1/PD-L1 immune therapy-acquired drug-resistant mouse based on in vivo multidimensional immune landscape evolution according to claim 1, wherein in the step S5, the analysis index of the immune microenvironment characteristics at least comprises the proportion change of dendritic cell DC, granulocyte Granulocyte, macrophage Macrophage and monocyte Monocyte and the proportion change of CD8+T cells.
  6. 6. The method for constructing a model of subcutaneous tumor in mice with acquired drug resistance by PD-1/PD-L1 immunotherapy based on in vivo multidimensional immune landscape evolution according to claim 1, wherein in step S1, the cell culture medium is DMEM high sugar medium containing 10% fetal bovine serum and 1% green-streptomycin, and the cell inoculation concentration is adjusted to 1x10 x 7cells/mL.
  7. 7. The method for constructing a subcutaneous tumor model of a PD-1/PD-L1 immune therapy acquired drug resistant mouse based on in vivo multidimensional immune landscape evolution according to claim 1, wherein in the step S2, in the construction of the subcutaneous tumor mouse model, the mouse is a C57BL/6 strain, an injection site is inguinal subcutaneous, the concentration of injection cell suspension is 1x10≡7cells/mL, and the injection volume is 0.1mL.
  8. 8. The method for constructing the subcutaneous tumor model of the PD-1/PD-L1 immune therapy acquired drug resistant mice based on in-vivo multidimensional immune landscape evolution according to claim 1, wherein the peripheral blood sampling in the step S4 adopts an orbital vein blood sampling method, the single blood sampling amount is controlled to be 100-200 mu L, and EDTA is used for anticoagulation.

Description

PD-1/PD-L1 immune treatment acquired drug-resistant mouse subcutaneous tumor model construction method based on in-vivo multidimensional immune landscape evolution Technical Field The invention relates to the field of animal model construction, in particular to a method for constructing a PD-1/PD-L1 immune therapy acquired drug-resistant mouse subcutaneous tumor model based on in-vivo multidimensional immune landscape evolution. Background PD-1/PD-L1 immunotherapy has revolutionary efficacy in some cancers, but acquired resistance is a major clinical bottleneck limiting its long-term efficacy. Acquired drug resistance is a dynamic evolutionary process involving complex remodeling of tumors and immune microenvironments under therapeutic pressure. Parsing this process and finding predictive markers is critical to developing synergy strategies. The existing preclinical model has the defects that a drug-resistant cell line induction model is disjointed with the in-vivo microenvironment, an in-situ recurrence model has long period and fixed immune background, and the immune system of a humanized mouse model is incompletely reconstructed. These models are all end-point studies, lacking a continuous, multidimensional assessment system for the overall process of "susceptibility to drug resistance" in the same individual. Thus, there is a need to construct a standardized, reproducible, dynamically monitorable preclinical model. Therefore, the invention provides a method for constructing a PD-1/PD-L1 immune therapy acquired drug-resistant mouse subcutaneous tumor model based on in-vivo multidimensional immune landscape evolution. Disclosure of Invention The invention aims to solve the defects in the prior art, and provides a PD-1/PD-L1 immunotherapy acquired drug-resistant mouse subcutaneous tumor model construction method based on in-vivo multidimensional immune landscape evolution. In order to achieve the above purpose, the present invention adopts the following technical scheme: The method for constructing the PD-1/PD-L1 immune treatment acquired drug-resistant mouse subcutaneous tumor model based on in-vivo multidimensional immune landscape evolution is characterized by comprising the following steps of: s1, preparing tumor cells, namely resuscitating and amplifying MC38 colon adenocarcinoma cell lines to prepare single-cell suspension; S2, establishing a basic model, namely inoculating MC38 single cell suspension to the subcutaneous of a homologous C57BL/6 mouse, and establishing a tumor-bearing mouse model; S3, drug resistance induction and screening, namely, after the tumor grows to a preset volume, applying continuous treatment pressure of an anti-PD-1 or anti-PD-L1 antibody to the tumor-bearing mice, firstly, comparing the tumor volume of the mice in the treatment group with the average volume of a control group at the same time point, screening out a 'response mouse' with the tumor volume obviously smaller than that of the control group, and eliminating primary drug resistant individuals; S4, dynamic immunity monitoring sampling, namely, in the treatment process of the step S3, non-lethal peripheral blood collection and tumor biopsy sampling are carried out on the same mouse individual according to a preset time point; s5, sample treatment and analysis, namely preparing the sample obtained in the step S4 into single-cell suspension, analyzing the immune microenvironment characteristics through flow cytometry, and verifying the drug resistance phenotype. Preferably, in the step S3, the specific scheme of drug resistance induction is that drug administration is started when the subcutaneous tumor volume reaches 50-100mm < 3 >, the dosage and frequency of drug administration are 5-10mg/kg or 200 mug/dose of anti-PD-1 antibody by intraperitoneal injection, the frequency is once every 3 days, the drug administration is continued until the drug resistance judgment end point is reached, and the ethical end point is judged when the tumor volume reaches 2000mm < 3 >. Preferably, in the step S3, the judgment standard of the acquired drug resistance model is simultaneously under the condition that initial response confirms that the tumor volume of the mice regresses or the disease is stable at the initial stage of treatment and the volume is obviously lower than that of a control group, drug resistance progress confirms that the tumor volume continuously increases by 3 times under the continuous administration condition, the volume threshold value is that the tumor volume rebounds to be 5 times higher than the initial volume, and the tumor volume is monitored by using a vernier caliper, and the calculation formula is that the tumor volume = 1/2 length x width square. Preferably, in the step S4, the tumor biopsy sampling adopts a biopsy needle puncture or minimally invasive surgery method, and 5-20mg of tumor core tissue is obtained on the premise of not killing the mice. Preferably, in the step S5, the analysis index