High-fat diet induces C-reactive protein secretion, promoting lung adenocarcinoma via immune microenvironment modulation

Figures

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  Fig. 1.. HFD promotes lung cancer progression in the mutant EGFR-driven transgenic mouse model. (A) Schematic of the experimental animal design. Six-week-old adult transgenic mice were randomly divided into four groups: LC-HFD, LC-RD, HFD and RD. LC-HFD and LC-RD mice had double transgenes (CC10-rtTA/Tet-O-EGFRL858R). They were fed an HFD or RD for 2 weeks and then switched to HFD+Dox or RD+Dox, respectively, for 8 weeks. HFD and RD mice had a single transgene (CC10-rtTA). They were fed an HFD or RD for 2 weeks and then switched to HFD+Dox or RD+Dox, respectively, for 8 weeks. Mutant EGFR-driven lung cancer was induced by Dox. (B) Representative photographs of mice at the end of the experiments. Scale bars: 2 cm. (C) Growth curve of mice in the HFD and RD groups (n=6 for each group). (D) Growth curve of mice in the LC-HFD and LC-RD groups (n=6 for each group). (E) Representative photographs of mouse lungs from each group. Scale bars: 1 cm. (F) Lung weight measurements of mice from each group at the end of the experiments. Results are shown as mean±s.d. Two-way ANOVA was used for statistical analysis in C; unpaired two-tailed Student's t-test was used for statistical analysis in D and F.…

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  Fig. 2.. Histopathological analysis and molecular characterization of lung tumor tissues in mice. (A) Top panel: representative H&E staining of lungs from each group of mice at the end of the experiments. Scale bars: 100 μm (100×); 25 μm (400×). Bottom panel: quantification of tumor burden in LC-HFD and LC-RD mice (n=9 for each group). (B) Top panel: immunohistochemical analysis of EGFR and TTF-1 for evaluating tumor burden. Scale bars: 25 μm. Bottom panel: quantification of EGFR- and TTF-1-positive area in LC-HFD (n=5) and LC-RD (n=3) mice. (C) Western blot analysis of EGFR expression and activity. The quantified protein levels relative to GAPDH internal control are indicated below bands. (D) Quantification of Ki67 mRNA levels for evaluating proliferative tumor cells in the LC-HFD and LC-RD groups (n=4 for each group). Results are shown as mean±s.d., and unpaired two-tailed Student's t-test was used for statistical analysis. **P<0.01; ***P<0.001.

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  Fig. 3.. Whole-genome transcriptomic analysis and regulatory network construction for HFD-induced lung cancer progression. Total RNAs from three independent mouse lungs of the LC-HFD and LC-RD groups were reverse transcribed to cDNA and subjected to cDNA expression microarray experiment and analysis. (A) Principal component analysis of the global gene expression data from the LC-HFD and LC-RD groups (n=3 for each group). (B) Left panel: gene set enrichment analysis of whole-genome transcriptomic profiling using the cDNA expression microarray. Highly significantly enriched gene sets in LC-HFD group are shown. The green curve represents the density of the genes identified in the microarray. The FDR was calculated by comparing the actual data with the output of 100 Monte-Carlo simulations. The NES describes the density of modified genes in the dataset assuming random distribution. Right panel: heat maps of significantly enriched genes in each hallmark in the LC-HFC group, including IFN-α response, IFN-γ response, complement and IL-6/JAK/STAT3 signaling. (C) Volcano plot of differentially expressed genes (LC-HFD versus LC-RD). Blue dots represent genes that were significantly downregulated…

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  Fig. 4.. Identification of cytokines and adipokines potentially contributing to HFD-induced lung cancer progression. Mouse lung tissue was used to profile a total of 62 cytokines, and the serum was used to profile a total of 38 adipokines through antibody arrays. IPA software was used to further analyze the results on regulatory network prediction and the correlation of clinical outcomes through KM plotter analysis. (A) Left panel: a cytokine antibody array was prepared using lung cancer tissues from LC-HFD and LC-RD mice. The red and blue boxes represent upregulated and downregulated cytokines, respectively, in the LC-HFD compared with LC-RD group. Right panel: quantification of cytokines using ImageJ software. (B) Left panel: the levels of 38 different adipokines in mouse sera. The yellow boxes represent CRP upregulated in the LC-HFD group compared with that in the LC-RD group. Right panel: quantification of adipokines using ImageJ software. (C) Establishing the potential regulatory network underlying cytokine and adipokine modulation in HFD-treated mice with lung cancer using IPA software. (D) Quantitative real-time PCR analysis of Crp mRNA expression levels in the liver and adipose…

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  Fig. 5.. CRP enhances lung cancer cell proliferative ability and invasiveness. (A-C) Evaluation of cell proliferation through MTS assay of CRP-treated CL1-0 (A), PC9 (B) and H3255 (C) lung adenocarcinoma cells (top panel), and quantification of relative proliferative abilities (bottom panel). (D-F) Evaluation of cell migration through wound-healing assays of CRP-treated CL1-0 (D), PC9 (E) and H3255 (F) lung adenocarcinoma cells (left panel), and quantification of relative migratory abilities (bottom panel). Dashed lines in D-F indicate the initial cell boundary. (G-I) Evaluation of cell invasiveness through Matrigel transwell assays of CRP-treated CL1-0 (G), PC9 (H) and H3255 (I) lung adenocarcinoma cells (left panel), and quantification of invaded cells (right panel). Scale bars: 20 μm. Results are shown as mean±s.d., and unpaired two-tailed Student's t-test was used for statistical analysis. ns, not significant; *P<0.05; **P<0.01; ***P<0.001. OD, optical density.

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  Fig. 6.. HFD-induced CRP secretion and lung cancer progression. HFD increases CRP secretion from the liver and adipose tissue in mice with lung cancer. Circulating CRP modulates immune responses and induces inflammation in the tumor microenvironment. CRP stimulates tumor cell proliferation, promoting lung cancer progression.