Gene expression profiling is a powerful approach to address the mechanism of action (MOA) of cancer immunotherapy-driven reprogramming of tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment. However, TILs, particularly T and NK cells, are a very rare population within the tumor. Gene expression profiling using Nanostring or RNAseq is often not sensitive enough to detect expression changes in rare populations, and it is difficult to separate gene expression profiles between different cell subsets. Although single-cell RNAseq can distinguish gene expression profiles between different cell subsets, it is very costly and would require many cells to be sequenced before obtaining the rare cell type of interest. Classically, fluorescence-activated cell sorting (FACS) has been used to isolate rare populations, but this method is a long process that can affect cell phenotype and results in dramatic cell loss. Thus, we developed a protocol for rapid and reproducible magnetic bead-based isolation of tumor-infiltrating lymphocytes (TILs) for higher sensitivity MOA studies. CD8 and CD4 TILs were isolated from both MC38 and CT26.KSA tumor models with high purity and sufficient yield for further Nanostring gene expression analysis. As expected, CD8 and CD4 T cell gene expression profiles were dramatically enriched in the isolated TILs compared to the whole tumor. Thus, our protocol for isolating TILs from mouse tumors can be applied broadly to multiple tumor types and provides sufficient yield to perform downstream high-sensitivity gene expression profiling.
Citation Format: Lindsay M. Webb, Bartholomew Naughton, Sireesha Yalavarthi, Clotilde Bourin, Jacques Moisan, Chunxiao Xu. Isolation of tumor-infiltrating lymphocytes for higher sensitivity gene expression profiling [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO062.