A practical workflow for isolation and low-input multi-omic and functional profiling of neutrophils from tumor tissue

Tumor-associated neutrophils (TAN) are increasingly recognized as major regulators of tumor progression, metastasis, immune suppression, and response to immunotherapy. However, mechanistic studies of TAN remain technically challenging due to the frequently limited numbers recoverable from individual tumor specimens, susceptibility to activation during tissue processing, rapid apoptosis ex vivo , and poor survival following fluorescence-activated cell sorting (FACS). These limitations often compromise downstream molecular and functional analyses and contribute to substantial variability between laboratories. Importantly, the difficulty of recovering highly viable TAN has also limited direct functional validation of neutrophil states identified by transcriptomic profiling, leading many studies to infer functional heterogeneity primarily from transcriptional signatures. Here, we describe a practical workflow for the isolation of highly viable TAN from murine and human solid tumors. The protocol combines gentle tissue dissociation, sterile low-stress cell sorting conditions, post-sort handling, and downstream applications including single-cell RNA sequencing (scRNA-seq) with bioinformatic considerations, bulk RNA sequencing, CUT&RUN epigenomic profiling, mini suppression assays, ex vivo cultures, real time quantitative PCR and western blotting. Particular emphasis is placed on pre-analytical variables that critically affect TAN viability and on neutrophil-specific considerations for scRNA-seq quality control and interpretation. The workflow is designed to be adaptable to different tumor types and enables robust characterization of low-frequency neutrophil populations that are typically difficult to study using conventional approaches, facilitating experimental validation of the biological functions associated with transcriptionally defined TAN states.