In Vivo Immune Checkpoint Blockade Assay Protocol

Purpose

This protocol describes the steps to perform an in vivo immune checkpoint blockade assay, which is used to evaluate the efficacy of immune checkpoint inhibitors in promoting anti-tumor immunity in animal models.

Please note that this protocol is a general guideline, and modifications may be necessary depending on the specific experimental setup, animal model, and immune checkpoint inhibitors used.

Materials

- Mice (e.g., C57BL/6)

- Syngeneic tumor cell line (e.g., MC38 for C57BL/6 mice)

- Anti-PD-1, anti-CTLA-4, or other immune checkpoint inhibitor antibodies

- Phosphate-buffered saline (PBS)

- Syringes and needles

- Caliper for tumor measurement

- Isoflurane or other anesthetic agent

Procedure

1. Inoculate syngeneic tumor cells (e.g., 5 × 10^5 MC38 cells) subcutaneously into the right flank of mice.

2. Allow tumors to establish and reach a palpable size (approximately 50-100 mm^3), which typically takes 7-10 days post-inoculation.

3. Randomize the mice into treatment groups (e.g., control, anti-PD-1, anti-CTLA-4, or combination therapy).

4. Administer immune checkpoint inhibitor antibodies or control (e.g., isotype control) intraperitoneally at the desired dose and schedule. A common dosing regimen is 200 μg per mouse every 3-4 days.

5. Monitor tumor growth by measuring tumor size with a caliper every 2-3 days. Calculate tumor volume using the formula: volume = (length × width^2) / 2.

6. Continue treatment and tumor measurements for a predetermined period (e.g., 2-4 weeks) or until tumors reach a predefined endpoint size (e.g., 1,500 mm^3).

7. Euthanize the mice at the end of the study or when tumors reach the endpoint size.

8. Collect tumors and other relevant tissues (e.g., draining lymph nodes, spleen) for further analysis, such as flow cytometry, histology, or gene expression profiling.

Data Analysis

1. Calculate the mean and standard error of tumor volumes for each treatment group at each measurement time point.

2. Plot tumor growth curves showing the mean tumor volume ± standard error over time for each treatment group.

3. Perform statistical analysis (e.g., two-way ANOVA with repeated measures) to determine if there are significant differences in tumor growth between treatment groups.

4. If the immune checkpoint inhibitor-treated groups show significantly delayed tumor growth or reduced tumor volumes compared to the control group, it indicates that the therapy is effective in promoting anti-tumor immunity.

Notes

• The choice of syngeneic tumor model and mouse strain may depend on the specific research question and the immune checkpoint inhibitor being tested.
• The dosing regimen and schedule of immune checkpoint inhibitors may need to be optimized based on previous studies or pilot experiments.
• Additional analyses, such as flow cytometry to characterize immune cell populations or histology to assess tumor infiltration, can provide further insights into the mechanisms of action of the immune checkpoint inhibitors.
• Proper animal care and handling practices should be followed, and all experiments should be conducted in accordance with institutional, national, and international guidelines for animal welfare and ethics.

• When injecting antibodies into mice, the choice of diluent is crucial for ensuring the stability of the antibodies and the safety of the animals. Here are common diluents used:

   

  • Phosphate-buffered saline (PBS): This is a widely used buffer because it is isotonic and non-toxic to cells. PBS helps maintain pH and osmolarity, which is essential for in vivo applications.

  • Normal saline: Saline is another common choice, particularly for in vivo applications where buffering is not as critical. It is isotonic and helps maintain the physiological balance of the cells.

  • Other buffer systems: Depending on the specific application and the properties of the antibody, other buffer systems such as Tris-buffered saline (TBS) may be used.

• It's essential to check the compatibility of these buffers with both the antibodies and the animal model.

• Always ensure the solution is sterile and free from pyrogens, endotoxins, or preservatives such as sodium azide, which could be harmful to the mice. It's also crucial to use a buffer that maintains the antibody's integrity and functionality without causing harm or stress to the animal.