Abstract
In recent years, research and development of immunity checkpoint inhibitors such as anti-PD-1 antibody and anti-CTLA-4 antibody have become active. Therefore, a model that can evaluate immunity checkpoint inhibitors is required.
In this study, we examined the evaluation method for tumor infiltrating lymphocytes (TIL), which is one of the evaluations of immunity checkpoint inhibitors, in addition to conventional antitumor evaluation using CT26WT-bearing mice.
Mouse colorectal cancer cell line CT26WT was transplanted subcutaneously into BALB/c mice and sorted into the control group and drug treatment group based on the tumor volume (Day 0). Anti-PD-1 antibody or control antibody was administered intraperitoneally on Day 7 and 14 and observation was carried out until Day 21. During the experimental period, the tumor diameter was measured to evaluate the antitumor effect of the drug. Furthermore, the tumor was excised and dispersed by gentleMACS dissociator (Miltenyi Biotec K.K.). Isolation of TILs was performed using CD45 microbeads for magnetic cell sorting, based on MACS Technology. Antibody staining was performed to the isolated CD45 positive cells, and TIL analysis was performed by flow cytometer, BD FACSLyric (Becton, Dickinson and Company).
As a result, regulatory T cells (CD4+/CD25+/FoxP3+) isolated from tumor cells were observed like those in the spleen used as a control. Changes in lymphocyte distribution were observed in the drug treatment group.
These results indicated that by using the current protocol and measurements, an evaluation system for TIL has been successfully established in the CT26WT-bearing mice model.
