Fig 1: Epigenetically imprinted lung microenvironment by viral infection promotes lung tumor growth(A) Schematic of experimental setup for bone marrow transplantation and tumor implantation.(B) Representative image of tumor-bearing lung lobes (left) and quantification of tumor burden (right) at week 3 in PBS- or IAV-bone marrow transferred KPi.t. mice.(C) Tumor growth curves of PBS- or IAV-infected mice s.c. implanted with KP tumor cells.(D) Schematic of the tumor parabiosis experiment. Wild-type female CD45.1+ and CD45.2+ congenic mice were treated with PBS or infected with IAV, respectively, and maintained for 4 weeks prior to surgical parabiosis (forming PBS-IAV pairs). PBS-PBS pairs served as controls. After 3 weeks of parabiosis recovery, both mice in each pair were i.t. inoculated with KP tumor cells. Tumor burden and neutrophil populations were analyzed 3 weeks later.(E and F) Frequency and cell number of SiglecFhi neutrophils in the lung parenchyma (E) and blood (F) among parabiosis pairs.(G) Frequency and cell number of SiglecFhi neutrophils in the lung parenchyma of parabiosis pairs, stratified by CD45.1+ and CD45.2+ origin to distinguish host- vs partner-derived cells.(H and I) Representative image of tumor-bearing lung lobes (H) and quantification of tumor burden (I) at week 3 among parabiosis pairs.(J) Heatmap showing upregulated cytokines and chemokines in BAL from IAV_Cre, IAV_KPi.t. and SCV2_KPi.t. mice compared to their respective PBS-treated controls.(K) Representative scATAC-Seq genomic browser tracks showing chromatin accessibility at the Csf3 locus in AM, IM, epithelial cells, fibroblasts and endothelial cells from PBS- and SCV2-infected lungs at 28 d.p.i.(L) Representative bulk ATAC-Seq genomic browser tracks displaying chromatin accessibility at the Csf3 locus in AM isolated from BAL of PBS- and IAV-infected mice at 35 d.p.i.(M) G-CSF production by SCV2- or IAV-infected lung cells or AMs at 35 d.p.i. following overnight stimulation with LPS or UV-irradiated KP cells. Culture supernatants were collected and analyzed by ELISA.(N) Flow cytometry dot plots showing the induction of SiglecF+ neutrophils following recombinant G-CSF or TGF-β treatment, as well as G-CSF/TGF-β and GM-CSF/TGF-β co-treatment (left), and the proportion of SiglecF+ neutrophils (right).(O) Blockade of G-CSF receptor (G-CSFR) reduced both the frequency and number of SiglecFhi neutrophils in tumor-bearing lungs of mice previously infected with IAV.(P) Representative image of tumor-bearing lung lobes and quantification of tumor burden from (O).(Q) Schematic model illustrating how prior severe respiratory viral infection epigenetically reprograms lung microenvironment via G-CSF production, promoting the local accumulation of SiglecFhi neutrophils and contributing to accelerated tumor growth.Data represent two independent experiments or pooled from two experiments. Graphs display mean ± SEM. Statistical significance was assessed by Mann-Whitney test (B and J), two-way ANOVA (E, F, G, and I), and one-way ANOVA (N). (ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)