BackgroundRUNX3　acts　as　a　tumor　suppressor　gene　in　non-small-cell　lung　cancer　(NSCLC),　yet　its　specific　biological　mechanism　is　still　unclear.　This　study　aimed　to　uncover　tumor　microenvironment　(TME)　changes　in　NSCLC　with　varying　RUNX3　expression　statuses　through　single-cell　RNA　sequencing.Patients　and　MethodsIn　total,　seven　patients　with　NSCLC　with　detailed　pathological　data　were　involved,　with　three　both　paracancerous　and　cancerous　tissue　samples.　After　sequencing,　the　＂Seurat＂　package　was　used　to　analyze　differentially　expressed　genes,　annotate　cell　clusters　with　marker　genes,　and　compare　cell　proportion　differences　at　different　RUNX3　expression　levels.　Observed-over-expected　cell　number　ratios　(Ro/e)　assessed　cell　type　enrichment　among　three　pathological　types.ResultsImmunohistochemical　staining　of　RUNX3　categorized　three　patients　into　the　RUNX3-negative　group　(RUNX3_Neg)　and　four　into　the　RUNX3　positive　group　(RUNX3_Pos).　All　cells　were　classified　into　13　types　based　on　marker　genes.　Ro/e　results　showed　fibroblasts　were　the　only　enriched　cell　type　in　RUNX3_Pos　cancer　tissue,　while　club　cells,　ciliated　cells,　and　so　on　were　enriched　in　RUNX3_Neg　cancer　tissue.　RUNX3_Neg　tissues　were　more　likely　to　accumulate　certain　immune　cells　compared　with　RUNX3_Pos　tissues.　Ro/e　also　indicated　RUNX3_Neg　cancer　tissues　were　more　prone　to　macrophage　depletion,　while　RUNX3_Pos　tissues　were　more　prone　to　macrophage　enrichment.ConclusionsThrough　single-cell　sequencing,　our　study　found　that　RUNX3　expression　status　is　closely　related　to　NSCLC　TME.　Mononuclear　phagocytes　may　be　an　important　target　cell　population　for　RUNX3　to　change　TME.