Exosomal POSTN from cancer-associated fibroblasts drives progression of microinvasive lung adenocarcinoma: insights from single-cell and tissue exosome sequencing analysis

Figure 1. Flow chart of this study.

Figure 2. Single-cell transcriptomic landscape of MIA and matched adjacent lung tissues. (A) UMAP visualization of all cells colored by unsupervised clusters and by sample origin. (B) UMAP plot showing the annotation of major cell types, including B cells, dendritic cells, endothelial cells, epithelial cells, fibroblasts, macrophages, mast cells, neutrophils, NK cells, plasmacytoid dendritic cells (pDCs), plasma cells, and T cells. (C) Dot plot showing the expression of canonical marker genes used for cell-type annotation. (D) Stacked bar plot showing the relative proportions of major cell populations across individual samples.

Figure 3. Epithelial cell heterogeneity and identification of the malignant Cancer-AT2 subpopulation in MIA. (A) Epithelial cells were divided into 6 subsets, including Cancer-AT2, Normal-AT2, AT1, Club, Ciliated, Basal. (B) Markers for epithelial cell subsets. (C, D) Summary of epithelial cell subset proportions in carcinoma and adjacent tissues. (E, F) CNV analysis shows that the Cancer-AT2 cell population has the highest malignancy level among these subsets. (G) Feature plots of representative epithelial marker genes. (H) Violin plots showing subtype-specific expression of representative marker genes.

Figure 4. Heterogeneity of fibroblast-lineage cells in MIA and enrichment of POSTN+ CAFs in tumor tissues. (A) Fibroblasts were divided into 7 subsets: POSTN+CAFs, SFRP2+CAFs, VEGFD+CAFs, iCAFs, myoCAFs, EnMTCAFs, Pericyte. (B) Canonical marker genes for fibroblast subtype annotation shown as a dot plot. (C) Comparison of subtype-associated marker expression between cancer and adjacent tissues. (D, E) Summary of fibroblasts cell subset proportions in carcinoma and adjacent tissues. (F) Feature plots of representative marker genes across fibroblast-lineage cells. (G) Violin plots showing subtype-specific expression of representative marker genes.

Figure 5. Integrated analysis of tissue-derived exosomes and single-cell transcriptomes identifies fibroblasts as the cell population most closely associated with exosomal signatures in MIA. (A) Heatmap showing the enrichment of exosome-related pathways across major cell populations. (B) Bubble plot showing the correlations between major cell populations and tissue-derived exosomal transcriptomic profiles. (C) Pairwise overlap matrix of highly expressed genes among major cell populations. (D) Dot plots showing the expression of representative macrophage-associated genes, neutrophil-associated genes, and T-cell-associated genes across major cell populations. Dot size represents the percentage of cells expressing each gene, and color intensity indicates the average expression level.

Figure 6. Enhanced epithelial–fibroblast crosstalk centered on cancer-AT2 cells and POSTN+ CAFs in MIA. (A) Bar plots showing the total number of inferred interactions and the overall interaction strength between epithelial and fibroblast compartments in cancer and adjacent tissues. (B) Differential interaction network among epithelial and fibroblast subpopulations. (C) Heatmaps showing overall signaling patterns of epithelial and fibroblast subpopulations in cancer and adjacent tissues. (D) Heatmaps showing incoming signaling patterns of epithelial and fibroblast subpopulations in cancer and adjacent tissues. (E) Molecules unique to tumor or adjacent tissue in epithelial-fibroblast communication.

Figure 7. Clinical evaluation of exosome-derived candidate genes and validation of POSTN in LUAD. (A) Expression levels of the five most upregulated differentially expressed mRNAs—CCL19, NHS, URB1/KIAA0539, PHLDA2, and POSTN—in tissue-derived exosomes from LUAD tissues and matched adjacent normal tissues. (B) Expression levels of CCL19, NHS, URB1, PHLDA2, and POSTN in tumor and normal lung tissues from the TCGA-LUAD cohort. (C) Kaplan–Meier overall survival analysis according to POSTN expression in LUAD. (D) Receiver operating characteristic (ROC) curves showing the diagnostic performance of the five candidate genes in LUAD. (E) Relative POSTN mRNA expression in BEAS-2B bronchial epithelial cells and LUAD cell lines (A549, H1299, HCC827, and PC9). (F) Relative POSTN mRNA expression in adjacent and LUAD tissues, and in non-metastatic and metastatic LUAD samples. (G) Tissue-derived exosomal POSTN mRNA levels in LUAD, stratified by lymph node metastasis status and pathological stage. (H) Relative serum exosomal POSTN mRNA expression in healthy controls and patients with LUAD. *Asterisks indicate statistical significance versus adjacent tissue or the non-metastatic group (**P < 0.01; ***P <…

Figure 8. In vitro and in vivo effects of POSTN+ CAF–derived exosomes on LUAD cells. (A, B) CCK-8 assays showing the proliferation of A549 (A) and H1299 (B) cells after treatment with exosomes derived from POSTN- CAFs or POSTN+ CAFs. (C) Representative images of colony formation in A549 and H1299 cells treated with exosomes from POSTN- CAFs or POSTN+ CAFs. (D, E) Representative images of Transwell migration and invasion assays in A549 (D) and H1299 (E) cells treated with exosomes from POSTN- CAFs or POSTN+ CAFs. (F) Tumor growth in nude mice bearing A549 or H1299 xenografts treated with PBS, POSTN- CAF–derived exosomes, or POSTN+ CAF–derived exosomes. *P < 0.05, **P < 0.01, ***P < 0.001 versus the control group; #P < 0.05, ##P < 0.01, ###P < 0.001 versus the POSTN- CAF–derived exosome group.