Establishment of an animal model of immune-related adverse events induced by immune checkpoint inhibitors

Abstract

Objective: Immunotherapy, specifically immune checkpoint inhibitors (ICIs), has revolutionized cancer treatment. However, it can also cause immune-related adverse events (irAEs). This study aimed to develop a clinically practical animal model of irAEs using BALB/c mice.

Methods: Subcutaneous tumors of mouse breast cancer 4T1 cells were generated in inbred BALB/c mice. The mice were treated with programmed death-1 (PD-1) and cytotoxic t-lymphocyte antigen 4 (CTLA-4) inhibitors once every 3 days for five consecutive administration cycles. Changes in tumor volume and body weight were recorded. Lung computed tomography (CT) scans were conducted. The liver, lungs, heart, and colon tissues of the mice were stained with hematoxylin-eosin (H&E) staining to observe inflammatory infiltration and were scored. Serum samples were collected, and enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of ferritin, glutamic-pyruvic transaminase (ALT), tumor necrosis factor-α (TNF-α), interferon-gamma (IFN-γ), and interleukin-6 (IL-6). Mouse liver and lung cell suspensions were prepared, and changes in macrophages, T cells, myeloid-derived suppressor cells (MDSCs), and regulatory (Treg) cells were detected by flow cytometry.

Results: Mice treated with PD-1 and CTLA-4 inhibitors showed significant reductions in tumor volume and body weight. The tissue inflammatory scores in the experimental group were significantly higher than those in the control group. Lung CT scans of mice in the experimental group showed obvious inflammatory spots. Serum levels of ferritin, IL-6, TNF-α, IFN-γ, and ALT were significantly elevated in the experimental group. Flow cytometry analysis revealed a substantial increase in CD3⁺T cells, Treg cells, and macrophages in the liver and lung tissues of mice in the experimental group compared with the control group, and the change trend of MDSCs was opposite.

Conclusions: The irAE-related animal model was successfully established in BALB/c mice using a combination of PD-1 and CTLA-4 inhibitors through multiple administrations with clinical translational value and practical. This model offers valuable insights into irAE mechanisms for further investigation.

Keywords: animal model; cancer immunotherapy; immune checkpoint inhibitors; immune‐related adverse events.

FIGURE 1

Construction of subcutaneously transplanted 4 T1 tumors in mice and establishment of an immune‐related adverse event (irAE) model. (A) BALB/c mice (n = 6/group) were subcutaneously injected with 4T1 tumor cells, and when the tumor reached a mean size of 30 mm³ (indicated by arrows), the tumor‐bearing mice were treated intraperitoneally with anti‐PD‐1 plus anti‐CTLA‐4 treatments or PBS once every 3 days for a total of five treatments. (B–D) The body weight, tumor growth, and tumor weight between the control group and aPD‐1 + aCTLA‐4 group. (E) The significant differences between body weight and tumor weight of mice. (F) The significant differences in spleen weight between the group of control and the group of aPD‐1 + aCTLA‐4. *p < 0.05; **p < 0.01.

FIGURE 2

Induction of immune‐related adverse event (irAE) by combination administration in 4T1‐bearing mice. Chest computed tomography (CT) was performed on the day before the last treatment, and liver, lung, heart, and colon tissues, and serum were collected. (A) Representative H&E staining sections of four tissues in the control and aPD‐1 + aCTLA‐4 groups, at 200× (left) and 400× (right). (B) Histological scores of H&E stained sections in two groups. (C) The images of lung CT for mice, the two images on the left were control group, and the two images on the right were aPD‐1 + aCTLA‐4 group. (D) Serum ferritin, IL‐6, IFN‐γ, TNF‐α, and ALT levels in the two groups. Each symbol represents one mouse. *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 3

Changes in immune cell populations in tissues in which immune‐related adverse event (irAE) occurred. Single‐cell suspensions of mouse liver and lung tissue were prepared for flow cytometry analysis. (A) Gating strategy for flow cytometry analysis. (B–E) The proportion of CD3⁺ T cells, F4/80⁺CD11b⁺ macrophages, CD4⁺CD25⁺FOXP3⁺ Treg cells, and CD11b⁺Gr‐1⁺ MDSCs. Each symbol represents one mouse. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not statistically significant.