Fig 1.
Representative 2D-DIGE profiling of differentially expressed proteins in the margin and center of ADC tumor.
(A) single-channel image of proteins from tumor margin, (B) single-channel image of proteins from tumor center. Red numbers indicate proteins that are more abundant in center and green numbers indicate proteins with more abundance in margin. Twenty-two spots for ADC (numbers correlate with descriptions in S1 Table) with significantly different abundance between the margin and center of tumor are shown (p < 0.05).
Fig 2.
Representative 2D-DIGE profiling of differentially expressed proteins in the margin and center of SCC tumor.
(A) single-channel image of proteins from tumor margin, (B) single-channel image of proteins from tumor center. Red numbers indicate proteins that are more abundant in center and green numbers indicate proteins with more abundance in margin. Twenty-one spots for SCC (numbers correlate with descriptions in S2 Table) with significantly different abundance between the margin and center of tumor are shown (p < 0.05).
Fig 3.
Immunoblotting verification of (A) mitochondrial aldehyde dehydrogenase 2 (ALDH2), (B) plastin (LCP1), (C) lamin 80 kDa (LMNA80 kDa) between center (Tc) and margin (Tm) of ADC tumor lung cancer and (D) pyruvate kinase PKM (PKM), (E) 40-kDa keratin (KRT19) and (F) rho GDP-dissociation inhibitor (ARHGDIB) between Tc and Tm of SCC tumor lung cancer. Results are expressed as means ± SD. Representative blots for one patient are shown. Asterisks indicate significant differences between Tc and Tm of tumor lung cancer (p < 0.05).
Table 1.
Top canonical pathways of differentially expressed proteins (DEPs) in ADC tumor.
Table 2.
Top canonical pathways of differentially expressed proteins (DEPs) in SCC tumor.
Table 3.
Top disease and disorder of differentially expressed proteins (DEPs) in ADC tumor.
Table 4.
Top disease and disorder of differentially expressed proteins (DEPs) in SCC tumor.
Table 5.
Molecular and cellular functions of differentially expressed proteins (DEPs) in ADC tumor.
Table 6.
Molecular and cellular functions of differentially expressed proteins (DEPs) in SCC tumor.
Fig 4.
Protein-protein interaction network of differentially expressed proteins in (A) margin vs. center, (B) center vs. control, (C) margin vs. control comparison for ADC. The network nodes represent proteins while the edges represent predicted functional associations. There are 5 types of associations presented: neighborhood (green), experimental (purple), text mining (yellow), database (light blue), coexpression (black) evidence. The color of the nodes represents cluster membership. Inter-cluster edges are depicted by dashed lines. The colored areas represent functional pathways.
Fig 5.
Protein-protein interaction network of differentially expressed proteins in (A) margin vs. center, (B) center vs. control, (C) margin vs. control comparison for SCC. The network nodes represent proteins while the edges represent predicted functional associations. There are 5 types of associations presented: neighborhood (green), experimental (purple), text mining (yellow), database (light blue), coexpression (black) evidence. The color of the nodes represents cluster membership. Inter-cluster edges are depicted by dashed lines. The colored areas represent functional pathways.
Fig 6.
(A) margin vs. center comparison in ADC. Cellular Movement, Hematological Disease, Immunological Disease Network is presented together with Signaling with Rho GTPases canonical pathway; (B) center vs. control comparison in ADC. Cellular Movement, Hematological Disease, Immunological Disease is presented together with Signaling with Epithelial Adherens Junction Signaling canonical pathway and (C) margin vs. control comparison in ADC. Cardiovascular Disease, Free Radical Scavenging, Small Molecule Biochemistry is presented together with Acute Phase Response Signaling canonical pathway.
Fig 7.
(A) center vs. margin comparison in SCC. Connective tissue disorders, post-translational modification, protein folding is presented together with HIF 1α signaling canonical pathway; (B) center vs. control comparison in SCC. Cell morphology, embryonic development, hair and skin development and function is presented together with glucocorticoid receptor signaling canonical pathway and (C) margin vs. control comparison in SCC. Cell morphology, embryonic development, hair and skin development and function is presented together with glucocorticoid receptor signaling canonical pathway.
Fig 8.
Venn diagrams and IPA comparison analysis for proteins in each NSCLC subtype, ADC and SCC.
(A) Venn diagrams comparing proteins identified in SCC or ADC for the following comparisons: center vs. margin, center vs. control, and margin vs. control. (B) The heat map of the top 20 Canonical Pathways (left panel) across different comparisons displaying the Fisher’s exact test p-value (expressed as–log[p-value]). Proteins positively or negatively correlated with Acute phase response signaling or Iron homeostasis signaling are shown as a protein expression fold change heat maps (right panel). (C) The heat map displays the z-scores (abs(z) ≥ 2 in at least on comparison) from top 20 Diseases and Functions analysis (orange and blue rectangles represent activation and suppression, respectively).
Fig 9.
Invasion of cells and cellular infiltration functions overlaid with proteins from the particular comparison for SCC and ADC.
(A) margin vs. control; (B) center vs. control. Each network shows the proteins in the particular analysis that have a causal or correlative relationship with the function and indicates how they might increase or decrease selected function. Legends are a graphical explanation of icons, lines and colors used in a graph. Difference in protein abundance is indicated below each protein.
Fig 10.
Combined protein-protein interaction network of differentially expressed proteins in center vs. margin comparison for both SCC (node red border) and ADC (node blue border).
The network nodes represent proteins while the edges represent predicted functional associations. There are 6 types of associations presented: neighborhood (green), co-occurrence (blue), experimental (purple), text mining (yellow), database (light blue), co-expression (black) evidence. The color of the nodes represents cluster membership. Inter-cluster edges are depicted by dashed lines. The colored areas represent functional pathways. HNRNPF = HNRPF.
Table 7.
Clinical and pathological characteristics of ADC and SCC patients whose samples were included in this study.
Fig 11.
Microscopic features of lung adenocarcinoma (figures A to B and D to E) following hematoxylin and eosin staining. (A) Whole slide image of tissue collected from the margin of tumor diagnosed as pulmonary adenocarcinoma. (B) Whole slide image of tissue collected from the center of tumor diagnosed as pulmonary adenocarcinoma. (C) Whole slide image of tissue collected from non-tumor tissue referred as normal tissue. (D) Region of interest: magnification 20x. Lung adenocarcinoma with mixed subtypes–sample collected from tumor margin. A tumor content of 90% was determined by the pathologist and can be seen in the slide scan. (E) Region of interest: magnification 20x. Lung adenocarcinoma with mixed subtypes–sample collected from tumor center. A tumor content of 90% was determined by the pathologist and can be seen in the slide scan. Dust-laden macrophages were marked with solid arrows. (F) Region of interest: magnification 20x. Lung parenchyma–sample collected from non-tumor tissue referred as normal tissue. Numerous hemosiderin-laden macrophages can be seen in this sample and the examples of these cells are marked with asterisks.
Fig 12.
Microscopic features of lung squamous cell carcinoma (figures A to B and D to E) following hematoxylin and eosin staining. (A) Whole slide image of tissue collected from the margin of tumor diagnosed as pulmonary adenocarcinoma. (B) Whole slide image of tissue collected from the center of tumor diagnosed as pulmonary adenocarcinoma. (C) Whole slide image of tissue collected from non-tumor tissue referred as normal tissue. (D) Region of interest: magnification 20x. Squamous cell carcinoma, large cell, non-keratinizing, tumor grade G2 –moderately differentiated–sample collected from tumor margin. A tumor content of 80% was determined by the pathologist and can be seen in the slide scan. Tumor cells are visible in this section part of the slide. The presence of tumor necrosis was determined by a pathologist during microscopic evaluation in 2% of the tissue section area. (E) Region of interest: magnification 20x. Squamous cell carcinoma, large cell, non-keratinizing, tumor grade G2 –moderately differentiated–sample collected from tumor center. A tumor content of 80% was determined by the pathologist and can be seen in the slide scan. The presence of tumor necrosis was determined by a pathologist during microscopic evaluation in 2% of the tissue section area. (F) Region of interest: magnification 20x. Lung parenchyma–sample collected from non-tumor tissue referred as normal tissue. A small number of hemosiderin-laden macrophages can be seen in this sample and these cells are marked with asterisks.