(A) Workflow for neutrophil isolation, RNA extraction, and RNAseq analysis. (B) Principal component analysis of 500 most varying genes in neutrophils isolated from blood, tumor, liver, and brain from mice with PDAC at 10 d.p.i., as well as blood from sham mice. (C) Venn diagram of different comparisons of transcripts expressed in neutrophils from different organs. (D) We identified putative ‘brain-specific’ transcripts by comparing the transcriptome of brain-infiltrating neutrophils to that of liver- and tumor-infiltrating neutrophils, as well as circulating neutrophils (all from tumor animals). In order to control for the nonspecific effects of malignancy on circulating neutrophils, we any excluded transcripts that were upregulated in circulating neutrophils from tumor animals compared to circulating neutrophils from sham animals. Using this approach, we identified 104 upregulated and 126 downregulated ‘brain-specific’ transcripts RLTV = regularized logarithm transformed value. (E) Heatmap of select brain-specific transcripts showing relative expression, comparing average of brain neutrophils to neutrophils in different organs. Functional enrichment analysis (based on Gene Ontology curation) of brain-specific transcripts identified enrichment for the term ‘extracellular space’ (GO:0005615) in upregulated genes and enrichment for the terms ‘external side of plasma membrane’ (GO:0009897), ‘immune response’ (GO:0006955), and”response to interferon-gamma’ (GO: 00034341) in downregulated genes. Several brain-specific upregulated transcripts encoded neutrophil granule components and enzymes, such as neutrophil granule protein (Ngp), the metalloproteinase ADAMTS5 (Adamts5) neutrophil elastase (Elane), lactoferrin (Ltf), cathelicidin antimicrobial peptide (Camp), and transthyretin (Ttr), as well as proteins important for granule secretion and NET formation such as dynamin 3 (Dnm3), synaptotagmin 15 (Syt15), Serpinb1a (Serpinb1a), Serpin Family E Member 2 (Serpine2), C/EBPε (Cebpe) (Gombart et al., 2003), and Myosin VIIA And Rab Interacting Protein (Myrip) (Desnos et al., 2003). We also observed an increase in genes for solute carriers (Slc gene family) and components of the Na/K ATPase, suggesting brain-infiltrating neutrophils are highly metabolically active. Many brain-specific downregulated transcripts encoded proteins important for immune function and responsiveness to T-cell-derived cytokines, such as MHC II (H2-Ab1), CXCL7 (Ppbp), PDLIM1 (Pdlim1, a negative regulator of NFκβ signaling), and the interferon-inducible genes Ifi203, Ifi205, and Ifi209.