Workflow for high-dimensional flow cytometry analysis of T cells from tumor metastases

We describe a multi-step high-dimensional (HD) flow cytometry workflow for the deep phenotypic characterization of T cells infiltrating metastatic tumor lesions in the liver, particularly derived from colorectal cancer (CRC-LM). First, we applied a novel flow cytometer setting approach based on single positive cells rather than fluorescent beads, resulting in optimal sensitivity when compared with previously published protocols. Second, we set up a 26-color based antibody panel designed to assess the functional state of both conventional T-cell subsets and unconventional invariant natural killer T, mucosal associated invariant T, and gamma delta T (gamma delta T)-cell populations, which are abundant in the liver. Third, the dissociation of the CRC-LM samples was accurately tuned to preserve both the viability and antigenic integrity of the stained cells. This combined procedure permitted the optimal capturing of the phenotypic complexity of T cells infiltrating CRC-LM. Hence, this study provides a robust tool for high-dimensional flow cytometry analysis of complex T-cell populations, which could be adapted to characterize other relevant pathological tissues.

Automated summary

This study describes a workflow for high-dimensional flow cytometry analysis of T cells infiltrating liver metastatic tumor lesions. By optimizing the flow cytometer settings, establishing a multi-color antibody panel, and accurately tuning the dissociation of samples, the phenotypic complexity of T cells infiltrating the tumor lesions was effectively captured. This study provides a robust tool for analyzing complex T cell populations and can be adapted to characterize other relevant pathological tissues.