Isolation Of Tumor-Infiltrating Leukocytes From Mouse Tumors

Abstract Cell-based immunotherapy is being evaluated in various types of cancer and it is one of the most rapidly growing and promising areas of cancer research. Tumor-infiltrating leukocytes (TILs) consist of highly diverse leukocyte subsets with major roles in cancer immune surveillance. Due to their relatively low frequency, tumor heterogeneity, and the abundance of tissue debris in tumor samples, it is difficult to isolate or analyze TILs with sensitivity and precision. To address this challenge, we have developed a simple method for isolating CD45+ TILs from mouse tumors. Performance was evaluated in three commonly used mouse models, namely the B16 melanoma, CT26 colon carcinoma, and 4T1 mammary tumor models. Solid tumors were induced by subcutaneous implantation of B16, CT26.WT, and 4T1 cancer cell lines into syngeneic recipients. Starting with a single-cell suspension, TILs from tumor samples were labeled with an antibody complex that links CD45+ cells to magnetic particles, then separated using an EasySep™ magnet. Using this method, TILs were enriched from 17.5 +/− 5.8% to 90.1 +/− 6.0% (n = 13) from B16 tumors, 27.9 +/− 9.4% to 74.2 +/− 12.3 % (n = 9) from CT26 tumors, and 39.5 +/− 8.0% to 87.5 +/− 3.7% (n = 6) from 4T1 tumors. The protocol can be easily modified to achieve higher purity or recovery as required by adjusting the addition volume of the antibody complex or particles. Importantly, major immune subsets including T cells, B cells, and myeloid cells are recovered after isolation. The EasySep™ Mouse CD45 TIL Isolation Kit allows researchers to isolate leukocytes from tumors with ease, improving the TIL downstream workflow. Furthering our understanding of TILs will be essential for developing effective immunotherapeutic strategies.