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. 2019 May 29;8:e45571. doi: 10.7554/eLife.45571

Figure 6. Airway and alveolar signatures in murine and human lung adenocarcinoma (LUAD).

(A, B) RNA of mouse urethane-induced LUAD cell lines, lungs obtained pre- and one week post-urethane treatment, airway epithelial cells (AEC), alveolar type II cells (ATII), and bone marrow-derived macrophages (BMDM) was examined by Affymetrix Mouse Gene ST2.0 microarrays (n = 4/group). (A) Heat map of genes significantly differentially expressed (overall ANOVA and FDR p<10−6) shows accurate hierarchical clustering. (B) Expression of the 30 top-represented transcripts of AEC, ATII, and BMDM in lungs and LUAD cells. See also Figure 6—figure supplements 1–4. (C, D) RNA of human LUAD (n = 40), never-smoker lung tissue (n = 30), primary AEC (n = 5), primary ATII (n = 4), and alveolar macrophages (AMΦ; n = 9) was analyzed by Affymetrix Human Gene ST1.0 microarrays. (C) Heat map of genes significantly differentially expressed (ΔGE > 5 fold) between LUAD and lung (ANOVA and FDR p<10−3) shows accurate hierarchical clustering. (D) Mean expression levels of the 30 top-represented transcripts of human AEC, ATII, and AMΦ in lungs and LUAD. (E, F) Gene set enrichment analyses, including normalized enrichment scores (NES), of mouse (E) and human (F) AEC, ATII, and BMDM/AMΦ signatures (defined as the top 1% expressed genes overall or exclusive to the cell type; n = 2) in mouse and human LUAD transcriptomes shows significant enrichment of the AEC (but not the ATII and BMDM/AMΦ) signature compared with lung (nominal p<0.0001 for all, family-wise error rates FWER <0.01). Gene symbols indicate the top three lagging genes from each signature and shows loss of Scgb1a1 (encoding CCSP) by LUAD. See also Figure 6—figure supplements 5 and 6. Data are given as violin plots. P, two-way ANOVA probabilities. ns, *, **, and ***: p>0.05, p<0.05, p<0.01, and p<0.001 for the indicated comparisons by Bonferroni post-tests. ANOVA, analysis of variance; FDR, false discovery rate.

Figure 6—source data 1. Cross-examination of signature genes of murine AEC, ATII cells, BMDM, LUAD cells and lungs.
elife-45571-fig6-data1.xlsx (8.8KB, xlsx)
DOI: 10.7554/eLife.45571.066
Figure 6—source data 2. Cross-examination of signature genes of human AEC, ATII cells, BMDM, LUAD cells and lungs.
elife-45571-fig6-data2.xlsx (8.7KB, xlsx)
DOI: 10.7554/eLife.45571.067
Figure 6—source data 3. Quantification of gene set enrichment analyses data shown in Figure 6E.
elife-45571-fig6-data3.xlsx (7.6KB, xlsx)
DOI: 10.7554/eLife.45571.068
Figure 6—source data 4. Quantification of gene set enrichment analyses data shown in Figure 6F.
elife-45571-fig6-data4.xlsx (7.6KB, xlsx)
DOI: 10.7554/eLife.45571.069

Figure 6.

Figure 6—figure supplement 1. Lineage-specific gene expression in mouse lung adenocarcinoma cell lines induced by urethane compared with mouse lungs.

Figure 6—figure supplement 1.

RNA of mouse urethane-induced lung adenocarcinoma (LUAD) cell lines, lungs obtained pre- and one week post-urethane treatment, and airway epithelial cells (AEC), alveolar type II cells (ATII), and bone marrow-derived macrophages (BMDM) was examined by Affymetrix Mouse Gene ST2.0 microarrays (n = 4/group). Shown is the number of genes out of the 30 top-represented transcripts of AEC, ATII, and BMDM within the top-2000-expressed genes of lungs and LUAD cells.
Figure 6—figure supplement 2. Loss of lineage marker expression in mouse lung adenocarcinoma cell lines induced by urethane.

Figure 6—figure supplement 2.

Mean expression levels of selected transcripts, including lineage markers and markers of histologic subtype in lung adenocarcinoma (LUAD) cell lines compared with lungs pre- and one week post-urethane treatment (A and B, microarrays from Figure 6—figure supplement 1, n = 2/group; C, qPCR, n = 3/group). AD, adenocarcinoma; SQ, squamous cell carcinoma; SC, small cell carcinoma. P, overall probability, two-way ANOVA. ****: p<0.0001 for the highlighted genes compared with lungs (red, significantly down-regulated; green, significantly up-regulated).
Figure 6—figure supplement 2—source data 1. Quantification of gene expression levels of data shown in Figure 6—figure supplement 2.
elife-45571-fig6-figsupp2-data1.xlsx (9.1KB, xlsx)
DOI: 10.7554/eLife.45571.070
Figure 6—figure supplement 3. Loss of lineage marker expression in mouse lung adenocarcinoma cell lines induced by urethane compared with mouse lungs: heat maps.

Figure 6—figure supplement 3.

528 genes differentially expressed between six different lung adenocarcinoma cell lines cultured from urethane-induced lung tumors and six benign respiratory mouse samples, including lungs of saline- and urethane-treated mice obtained at one week post-treatment, as well as primary mouse tracheal epithelial cells using the cut-offs indicated. Whole heat map (left) showing the accurate hierarchical clustering of the samples according to differentially expressed genes, as well as the top over- and under-represented genes (right). Note the universal loss of expression of lineage markers by lung adenocarcinoma cells (genes in red font). ANOVA, analysis of variance; FDR, false discovery rate.
Figure 6—figure supplement 4. Loss of lineage marker expression in mouse lung adenocarcinoma cell lines induced by urethane compared with mouse lungs: volcano plot.

Figure 6—figure supplement 4.

Shown are selected top over- and under-represented genes (arrows) from microarrays from Figure 6—figure supplement 2.
Figure 6—figure supplement 5. Mouse gene set enrichment analyses.

Figure 6—figure supplement 5.

Shown are gene set enrichment analyses of airway epithelial cell (AEC), alveolar type II cell (ATII), and bone marrow-derived macrophage (BMDM) transcriptome signatures in mouse lungs (top) and urethane-induced lung adenocarcinoma (LUAD) cell lines (bottom) transcriptomes. The data were used to design Figure 6E.
Figure 6—figure supplement 6. Human gene set enrichment analyses.

Figure 6—figure supplement 6.

Affymetrix Human Gene ST1.0 microarrays hybridized with RNA of human lung adenocarcinomas (LUAD; n = 40), never-smoker lung tissues (n = 30), primary airway epithelial cells (AEC; n = 5), primary alveolar type II cells (ATII; n = 4), and alveolar macrophages (AMΦ; n = 9) were cross-examined. Shown are gene set enrichment analyses of AEC, ATII, and AMΦ signatures in lung (top) and LUAD (bottom) transcriptomes. The data were used to design Figure 6F.