DOI: 10.25345/c5jd4q26m
title: MassIVE MSV000098823 - G-CSF/NAMPT signaling drives neutrophil dysfunction and enhances bacterial infection susceptibility in cancer patients
title: Despite advancements in cancer therapies, bacterial complications remain a major challenge, delaying treatment and worsening outcomes. While immunosuppressive therapies and prolonged hospitalizations contribute, they do not fully explain the elevated infection risk in cancer patients. We observed that elevated G-CSF production by tumors was associated with the persistence of Gram-negative pathogens in head and neck squamous cell carcinoma patients. The presence of Gram-negative pathogens in oral rinse positively correlated with poor prognosis of such patients. As neutrophils are the main cells controlling bacterial infections, we aimed to identify the molecular mechanisms underlying tumor-induced suppression of antibacterial activity of these cells. We identified tumor-driven activation of the NAMPT/NAD+ signaling axis in neutrophils (already at the progenitor level), which induced long-lasting cytoskeletal alterations, impairing phagocytosis and NETosis. Moreover, NAMPT/NAD+ activity accelerated neutrophil senescence and promoted accumulation of tissue-toxic CD62Llow subpopulations, ultimately disrupting lung tissue integrity and facilitating bacterial persistence in tumor-bearing hosts. Targeting the G-CSF/NAMPT signaling effectively reduced the generation of dysfunctional neutrophils and improved bacterial clearance in vivo. These findings reveal tumor-induced, NAMPT-dependent neutrophil reprogramming as a central mechanism driving impaired antimicrobial defenses. Implementing immune-modulating strategies, such as targeting G-CSF/NAMPT signaling, could improve infection control and enhance survival in cancer patients. Sample preparation: One mouse lung lobe (MOPC-bearing mice for 14 days or GCSF-producing-M4-mice bearing at 14 days) each was dissolved in 250 ul of lysis buffer (50 mM Tris (pH 7.8), 150 mM NaCl, and 5% SDS supplemented with complete mini-EDTA-free protease inhibitor, Roche, Penzberg). Samples were sonicated 2 times for 5 minutes on ice and centrifuged for five minutes at 16000g and room temperature to remove cell debris. A bicinchoninic acid (BCA) assay was performed (Pierce BCA Protein Assay Kit, (Thermo Fischer Scientific, Waltham, USA) to determine the protein concentration, following the manufacturers instructions. Tryptic digestion was performed using the single-pot, solid-phase-enhanced sample preparation (SP3) protocol, as described by Hughes et al. (2019). Peptides were dried in an Eppendorf concentrator plus (Eppendorf SE, Hamburg, Germany) and stored at minus 80 C until further use. Directly prior to measurement, dried peptides were resolved in 70 ul 0.1% FA and centrifuged at 16000g for 5 minutes to remove remaining beads. The supernatant was subjected to LC-MS analysis. LC-MS/MS data acquisition: Prior to injection, the peptide concentration was estimated using a NanoDrop Microvolume Spectrometer (Thermo Fischer Scientific, Waltham, USA) at 205nm. 300ng of peptides were loaded onto an Evotip (Evosep Biosystems, Odense, Denmark) following the manufacturers instructions. Peptide separation was performed on an Evosep One (Evosep Biosystems, Odense, Denmark) using the predefined Wisper 30SPD method on an PepSep Series column (C18, 15cm length x 150 um inner diameter, 1.5 um particle size (Bruker Daltronics, Brillerica, Massachusetts, USA)). Eluting peptides were injected into a TimsTOFpro mass spectrometer (Bruker Daltronics, Brillerica, Massachusetts, USA), via a CaptiveSpray source at 1600V. The mass spectrometer was operated in data-independent acquisition (DIA) PASEF mode. The accumulation and ramp time for the dual TIMS analyzer were set to 100ms at a ramp rate of 9.42 Hz. Scan ranges were set to m/z 100 -1,700 for the mass range and 1/k0 0.60 -1.60Vs-1cm-2 for the mobility range at both MS levels. For DIA-PASEF, a cycle time of 1.8 seconds was estimated. Within each cycle, a precursor mass range of 400 to 1200 Da and a mobility range of 1/K0 0.6 to 1.6 Vs-1cm-2 was fragmented. At MS1 level, one ramp was executed per cycle. For MS/MS measurements, 32 MS/MS windows with a mass width of 26.0 Da each and a mass overlap of 1.0 were distributed across the 16 MS/MS ramps. LC-MS/MS data processing: LC-MS raw data were processed using the directDIA algorithm in Spectronaut (Version 19.1.240806.626, Biognosys, Schlieren, Switzerland), applying BSG factory settings (Enzyme/ Cleavage rules: Trypsin/P; Fixed modification: Carbamidomethyl (C); Variable modifications: Acetyl (Protein N-term) and Oxidation(M)), against a reviewed mouse UniProt/Swissprot FASTA database (downloaded on 13th of March 2024, containing 17196 target sequences). Quantification was performed at the MS2 level. Imputation was disabled. Cross run normalization was enabled.