Fig 2: Illustrative overview of cellular compositions and spatial reorganization during LUAD invasion. SLPI‐expressiing AT2‐like2 cells could facilitate activation of the Wnt pathway, leading to elevated stemness and EMT, enhancing tumor cell invasiveness (top). Specific cellular subpopulations and their spatial localization that are proposed to contributed to invasion of LUAD (bottom).

Fig 3: Tissue‐resident macrophages establish a pro‐tumorigenic niche in LUAD tumors. A) UMAP plot showing five distinct clusters among 6142 macrophages from scRNA‐seq data, with colors representing cell types. B) Dotplot illustrating genes with differential expression patterns across five macrophage subtypes. C) Proportions of macrophage subtypes in MIA, IA and normal lung tissues from scRNA‐seq data. D) Boxplots displaying GSVA scores for each macrophage subtype in MIA and IA tumors based on scRNA‐seq data. Boxplot elements: center line, median; box limits, upper and lower quartiles; whiskers, 1.5 × IQR. Statistical significance assessed by two‐sided Mann‐Whitney U test. **** p < 0.0001. E) GO enrichment analysis of differentially expressed genes among five macrophage subtypes from SRT data. F, G, H) Inferred developmental trajectories of macrophage subtypes along pseudotime, distinguished by pseudotime progression(F), subtypes(G), and tissue types spanning MIA, IA and normal lung tissues from scRNA‐seq data (H). I) Proportion of three macrophage subtypes across different trajectory states in the scRNA‐seq data. J) Heatmap depicting differentially expressed genes among macrophage subtypes in the scRNA‐seq dataset, sorted according to pseudotime. K) IHC staining for MRC1 expression in tumor tissues from MIA and IA samples. Scale bar: 50 µm. L) IF staining for MRC1 (red) in tumor tissues from MIA and IA stages in LUAD patients. Nuclei were stained with DAPI (blue). Scale bar: 500 µm. M) Dotplot illustrating ligand‐receptor interactions between diverse cell types and receptors on RTM‐TAMs. Dot size reflects statistical significance (large dots: p < 0.01; small dots 0.01< p < 0.05), with only large dots shown. Analyses were performed using CellChat (v1.6.1) and CellChatDB.human (v1) database. N) Schematic overview of the protocol for isolating and sorting RTM‐TAM cells from mouse lung tissues. O) CBA assessment of cytokine levels in cell culture supernatants. LLC‐NC and LLC‐OE (SLPI‐overexpressing) cells were cultured alone, and LLC‐NC/LLC‐OE were co‐cultured with RTM‐TAM cells. Cytokine levels including IFN‐γ, IL‐6, IL‐10, IL‐12, MCP‐1, and TNF‐α were analyzed using CBA and flow cytometry. Statistical significance assessed by two‐way ANOVA test (n = 3). * p < 0.05, ** p < 0.01, ***p < 0.001; ns, not significant. IFN, interferon; IL, interleukin; MCP, monocyte chemoattractant protein; TNF, tumor necrosis factor.

Fig 4: SLPI contributes to invasiveness at the tumor margin. A) Spatial distribution of spots assigned to AT2 and AT2‐like subtypes (left panel) alongside gene‐set scores (right panel) in the IA stage from patient 7. Dotted line highlights an area enriched for AT2‐like 2 subtype at the tumor margin. B) Spatial distribution of spots based on gene‐set score in an IA sample from patient 8, featuring two tumors within a single section. Dotted lines illustrate areas enriched for AT2‐like 2 subtype near the tumor periphery. C) IF staining for SLPI and SOX2 in cells within the invasive region of IA‐stage LUAD samples. Dashed lines represent the border (n = 3). D) IF staining demonstrates elevated CD133 expression in tumor cells within the invasive region of IA‐stage LUAD samples (n = 3) (left: Low magnification, right: High magnification). Dash lines represent the border. E) IF staining revealing co‐expression of SLPI and SOX2 in tumor cells within the invasive region of IA‐stage LUAD samples. Dashed lines represent the border (n = 3). F) HE staining images of mouse lungs 8 weeks after tail‐vein injection of 2 × 106 PC9 cells (NC) and SLPI‐shRNA PC9 cells (sSLPI). Black outlines denote tumor formation regions (n = 6). G) The number of lung‐metastasis nodules on each lung surface. Data represents as mean ± standard deviation (S.D). Statistical significance assessed by unpaired t test (n = 6). ** p < 0.01. H) IF staining for E‐cadherin in xenografts excised from mice against (n = 3). Scale bar = 20 µm. I) WB analysis of CD133 and SOX2 expression in PC9 cells (NC) and SLPI‐shRNA PC9 cells (sSLPI) (n = 3). J) Differential gene expression patterns identified via RNA‐seq in PC9 Cells (NC) and SLPI‐shRNA PC9 Cells (sSLPI) (n = 3). K) WB analysis of Wnt/ pathway‐related indicators in PC9 cells (NC) and SLPI‐shRNA PC9 cells (sSLPI) (n = 3).