Histopathological grading and single-cell transcriptome profiles of mouse LUAD precancerous lesions.

A. Analysis and experimental flow chart of this study.

B. H&E images of normal tissue and three stages of precancerous lesions (AAH, adenoma, and AIS) in mice. Scale bar: 100 µm.

C. Immunofluorescence staining of ki67 at the four histopathological stages. Scale bar: 100 µm.

D. UMAP plot of 13 cell types from mouse scRNA-seq data.

E. Dotplot of marker genes in all cell types.

F. Cell distribution at the four stages.

G. Reduced transcriptional homogeneity with progression of precancerous lesions. Transcriptional heterogeneity between cells is inversely proportional to normalized mutual information (NMI).

AAH: atypical adenomatous hyperplasia; AIS: adenocarcinoma in situ; MIA: minimally invasive adenocarcinoma; IA: invasive adenocarcinoma; CM: conditioned medium; DC: dendritic cell; SMC: smooth muscle cell; ***p < 0.001.

Identification of initiation-associated epithelial marker Ldha and analysis of malignant epithelial cells.

A. UMAP plot of epithelial cell subtypes.

B. Dotplot showing the significance (P-value) and strength (communication probability) of specific interactions between epithelial cells with other cell types at the four stages. Data was obtained by cell chat analysis.

C. Immunofluorescence staining of Ldha at the four stages. Scale bar: 50 µm.

D. LDHA expression in LUAD and normal tissues from the TCGA database.

E. LDHA expression in stage I-IV LUAD and normal tissues from the TCGA database.

F. Correlation of LDHA expression with overall survival of LUAD patients in TCGA database.

G. Copy number variations (CNVs) (red, amplifications; blue, deletions) across the chromosomes (columns) inferred from the scRNA-seq of each epithelial cell (rows).

H. scMetabolism analysis of malignant epithelial cells at precancerous stages.

I. GSVA enrichment analysis of malignant epithelial cells at precancerous stages. LUAD: lung adenocarcinoma; *p < 0.05.

S100a4+ alv-macro was active in lipid metabolism at the AAH stage.

A. Malignant epithelial cells showed the strongest communication weight with alveolar macrophages, as shown by Cell chat analysis.

B. UMAP plot of S100a4 expression in alveolar macrophages.

C. scMetabolism analysis of S100a4+ alv-macro at the four stages.

D. Changes in scores of representative lipid metabolism-related gene sets across the four stages.

E. Compass analysis showing the reaction activities of fatty acid metabolism across the four stages.

F. Density plots of Cpt1a and Acot2 at the four stages. x-axis represents the gene expression level, and y axis represents the density of numbers of cells.

G. Multiplex immunofluorescence validation of F4/80+/Cd11c+/S100a4+ alv-macro in mouse normal and AAH tissues, and comparison of tissue expression of Cpt1a in this subpopulation. Scale bar: 20 µm.

The angiogenic function of S100a4+ alv-macro was related to fatty acid metabolism.

A. Cell proportion comparison of S100a4+ alv-macro in alveolar macrophages.

B. Macrophage functional program analysis of S100a4+ alv-macro across the four stages.

C. Pearson correlation analysis of angiogenesis and M2-like function with fatty acid metabolic reactions in S100a4+ alv-macro.

D. Density plots of Anxa2 and Ramp1 at the four stages.

E. Correlation analysis of Cpt1a and Anxa2 expression in S100a4+ alv-macro.

F. Multiplex immunofluorescence validation of the correlation between Cpt1a and Anxa2 expression in F4/80+/Cd11c+/S100a4+ alv-macro of mouse normal and AAH tissues. Scale bar: 20 µm.

S100a4-OE MH-S promoted malignant transformation of MLE12 epithelial cells in vitro.

A. S100a4 mRNA expression level in MH-S after plasmid transfection.

B. S100a4 protein expression level in MH-S after plasmid transfection.

C. CCK8 assay showing cell proliferation of MLE12 after coculture with S100a4-OE MH-S.

D. Colony forming ability of MLE12 after coculture, as shown by colony formation assay.

E. Cell cycle distribution of MLE12 after coculture, as shown by cell cycle analysis.

F. Wound healing assay showing cell migration of MLE12 after coculture.

G. Intracellular ROS level of MLE12 after coculture.

H. Transmission electron microscopy showing the morphological changes of mitochondria in MLE12 after coculture.

I. Western blotting of DNA damage marker p-γH2ax, EMT markers (E-cadherin, N-cadherin, and Vimentin), and stem-like markers (Cd44 and Cd133) in MLE12 after coculture.

J. Western blotting of tumorigenesis associated proteins (c-Myc, p-Erk, Sftpa, Vegfa, and Hif-1α) in MLE12 after coculture.

K. Western blotting of macrophage pro-tumor indicators (Vegfa, Mmp9, Tgf-β, and Hif-1α) in S100a4-OE MH-S.

L. Western blotting of fatty acid metabolism-related proteins (Cpt1a and Acot2) and angiogenesis-related proteins (Anxa2 and Ramp1) in S100a4-OE MH-S, and angiogenesis-related proteins (Anxa2 and Ramp1) in MLE12 after coculture.

OE: overexpression; NC: negative control; ROS: reactive oxygen species; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

S100A4+ alv-macro with similar pattern in human AAH stage

A. H&E images of five stages of human LUAD development (normal, AAH, AIS, MIA, and IA). Scale bar: 100 µm.

B. UMAP plot of subclusters of alveolar macrophages.

C. UMAP plot of S100A4 expression in alveolar macrophages.

D. Compass analysis of the reaction activities of fatty acid metabolism in S100A4+ alv-macro across the five stages.

E. Macrophage functional analysis of S100A4+ alv-macro across the five stages.

F. Dotplot of expression of CPT1A, ACOT2, and ANXA2 in the five stages.

G. Multiplex immunofluorescence staining of CPT1A and ANXA2 expression in CD68+/CD11C+/S100A4+ alv-macro of human normal and AAH tissues. Scale bar: 20 µm.