23 okt. 2025
Sadiku & Brenes et al. (BioRxiv)
Keywords
Neutrophils
Glioblastoma
Proteomics
Main Findings
Neutrophils in the tumour microenvironment (TME) are characterized by marked adaptability to systemic and intratumoral stimuli, having both pro- and anti-tumorigenic roles. The advent of single-cell transcriptomics has revealed new neutrophil subsets, but their functional significance remains unclear. Profiling the neutrophil proteome offers the potential to clarify their functional heterogeneity in the TME.
In this preprint (not peer reviewed), Sadiku, Brenes et al. describe a novel, high-throughput workflow for neutrophil proteomic profiling at single-cell resolution, using an ultra-high sensitivity mass spectrometer with a nanoflow liquid chromatography system. They analyzed FACS-sorted blood neutrophils and tumour-associated neutrophils (TANs) from 6 patients with glioblastoma (GBM) by mini-bulk proteomics, with TANs also profiled at single-cell resolution. This enabled the identification of more than 1,100 proteins per single TAN, while retaining deep coverage of granule and metabolic proteins crucial for neutrophil function.
Peripheral-blood analysis revealed expansion of an immature CD10⁻ neutrophil subset with mini-bulk proteomics showing variation within neutrophils to derive from maturity rather than density. CD10⁻ neutrophils had higher levels of ribosomal, mitochondrial and proliferation markers, whereas mature CD10⁺ cells were enriched for effector and inflammatory proteins. GBM TANs had a mitochondrial profile consistent with metabolic adaptation in the TME.
Single-cell profiling of TANs identified seven functional clusters. Of these, ‘armed’ and ‘engaged’ TANs were enriched for cytoskeletal and granule-associated proteins, consistent with motility and effector functions, whereas ‘exhausted’ cells had reduced metabolic and signalling capacity. Inferred trajectory analysis suggested a shift from ‘armed’ to ‘engaged’ states, diverging towards either ‘exhaustion’ or ‘vital neutrophil extracellular traps (NETs)’. ‘Lytic NET’ TANs had marked depletion of nuclear and granule components, whereas ‘immunosuppressive and angiogenic’ TANs upregulated proteins linked to phagocytic, immunosuppressive and pro-angiogenic activity. ‘Vascular immature’ neutrophils were scarce in tumours, consistent with limited infiltration or rapid maturation in the TME.
Novelty & Significance
This study provides the first single-cell proteomic characterization of human TANs in glioblastoma, revealing functional diversity not captured by transcriptomics. It introduces an ultra-sensitive workflow suited to low-protein immune cells and highlights neutrophil metabolic and effector plasticity within the GBM microenvironment.
Limitations
Limitations include a modest cohort, incomplete detection of low-abundance proteins, and the absence of spatial and temporal resolution needed to determine how neutrophil states are organized and evolve within the TME.
Reviewed by Aglaia Skolariki as part of a cross-institutional journal club between the Icahn School of Medicine at Mount Sinai, the University of Oxford, the Karolinska Institute, the MD Anderson Cancer Center, and the University of Toronto.
The author declares no conflict of interests in relation to their involvement in the review.