Diving Deep: Profiling Exhausted T Cells in the Tumor Microenvironment Using Spectral Flow Cytometry

Fresh tumor cytometric profiling is essential for interrogating the tumor microenvironment (TME) and identifying potential therapeutic targets to enhance antitumor immunity. Challenges arise due to the limited number of cells in clinical biopsies and inter-patient variability. To maximize data derived from a single biopsy, spectral cytometry was leveraged, enabling extensive profiling with significantly fewer cells than mass cytometry. Furthermore, the utilization of multiple markers within one tube can potentially reveal novel and extensive dynamic immune characteristics in cancer, thereby aiding treatment strategies and enhancing patient outcomes. Here, we introduce a customized 39-color panel for in-depth phenotyping of exhausted T cells (T_EX), which are dysfunctional T-cell subsets that arise during cancer progression. This study aims to investigate profiles of CD4 T, CD8 T, regulatory T (Treg), and γδ2 cells while exploring the heterogeneity of CD8⁺ T_EX subsets. Given the rarity and heterogeneity of tumor biopsies, we evaluated the effects of tissue dissociation enzymes on staining protocols using cryopreserved peripheral blood mononuclear cells (PBMCs). This is vital for the development of high-dimensional cytometry panels, especially since collagenases may cleave markers in dissociated tumor cells (DTCs). Our protocol also optimizes intracellular marker staining, enhancing insights into T_EX function and biology, ultimately identifying potential therapeutic targets.