Microbiome and metabolome analysis in smoking and non-smoking pancreatic ductal adenocarcinoma patients

Abstract

Background: Smoking is a significant risk factor for pancreatic ductal adenocarcinoma (PDAC). This study aimed to investigate the effects of smoking on the pancreatic microbiome and metabolome in resectable and unresectable male PDAC patients.

Methods: The pancreatic tissue samples were collected from resectable PDACs via surgery and unresectable PDACs via endoscopic ultrasound fine needle aspiration (EUS-FNA). Surgical samples obtained from 10 smoking and 6 non-smoking PDACs were measured by 16S ribosomal RNA (16S rRNA) gene sequencing and liquid chromatography-mass spectrometry (LC/MS). Fine needle aspiration (FNA) samples obtained from 20 smoking and 14 non-smoking PDACs were measured by 16S rRNA gene sequencing.

Results: From resectable to unresectable patients, the dominant genus in the pancreas changed from Achromobacter to Delftia. Smoking further altered the abundance of specific bacteria, mainly manifested as an increase of Slackia in surgical tumor tissue of the smoking group, and an enrichment of Aggregatibacter and Peptococcus in FNA samples of the smoking group. In tumor tissue, smoking caused an enrichment of the cancer-promoting cAMP signaling pathway and L-lactic acid. In paracancerous tissue, smoking also induced a detrimental disturbance in the pancreatic microbiome and metabolome, including an enrichment of Veillonella, Novosphingobium, Deinococcus, and 3-hydroxybutanoic acid, and a reduction of linoleic acid. Besides, the cancer-promoting L-lactic acid was negatively correlated with Faecalibacterium in tumor tissue based on the correlation analysis.

Conclusion: There were differences in the pancreatic microbiome of PDAC patients at different stages, and smoking can further disrupt the pancreatic microbiome and metabolism in PDAC.

Keywords: Endoscopic ultrasound-guided fine needle aspiration; Metabolome; Microbiome; Pancreatic ductal adenocarcinoma; Smoking.

Fig. 1

Comparison of the microbiome in tumor tissue of surgical samples from smoking vs. non-smoking PDAC. A, B Alpha diversity analysis of the smoking PDAC group (PDAC_smoking, n = 10) and non-smoking PDAC group (PDAC_non-smoking, n = 6). Box plots show the distribution of the Shannon and Sobs indices of alpha diversity scores. C Principal co-ordinates analysis (PCoA) quantified as weighted-unifrac dissimilarity. D Distinct bacterial composition at the genus level of subjects between the smoking PDAC group and non-smoking PDAC group. E Linear discriminant analysis (LDA) demonstrated distinct bacterial genera enriched in the smoking PDAC group and non-smoking PDAC group. Microorganisms with P < 0.05 and LDA score > 2 were considered significant. F Network analysis of the differential genera between the smoking PDAC group and non-smoking PDAC group. Only the genera with correlation coefficient > 0.5 and P < 0.05 were illustrated in the graph. The red line indicates positive correlation, while the green line indicates negative correlation. The size of the node indicates the abundance of the genus, and the thickness of the lines between nodes indicates the magnitude of the correlation

Fig. 2

Comparison of the metabolome in tumor tissue of surgical samples from smoking vs. non-smoking PDAC. A, B Pancreatic metabolic profile significantly differed between smoking PDAC and non-smoking PDAC group by OPLS-DA method. Test for the OPLS-DA model showed that the model for this study was valid. C Metabolites with significant differences between groups were presented in the volcano plot. Different metabolites were depicted by setting the fold-change > 1 and P < 0.05 as the threshold. The red points represented the upregulated metabolites (n = 18), and the blue points represented the downregulated metabolites (n = 25). D The altered signaling pathways in the smoking PDAC group compared with the non-smoking PDAC group by KEGG enrichment analysis, marked as * for P < 0.05. E Heatmap representative differential metabolites between the smoking PDAC group and non-smoking PDAC group. The color from blue to red indicates that the expression differential abundance of metabolites is from low to high. The right side shows the VIP bar chart of the metabolite, and the length of the bar represents the contribution value of the metabolite to the difference between the two groups. The color of the bar indicates the significance of the difference between the two groups of samples, P < 0.001 is marked as ***, P < 0.01 is marked as **, P < 0.05 is marked as *

Fig. 3

Comparison of the microbiome in paracancerous tissue of surgical samples from smoking vs. non-smoking PDAC. A, B, Alpha diversity analysis of the smoking PDAC group (PDAC_smoking, n = 10) and non-smoking PDAC group (PDAC_non-smoking, n = 6). Box plots show the distribution of the Shannon and Sobs indices of alpha diversity scores. C PCoA quantified as weighted-unifrac dissimilarity. D Distinct bacterial composition at the genus level of subjects between the smoking PDAC group and non-smoking PDAC group. E LDA demonstrated distinct bacterial genera enriched in the smoking PDAC group and non-smoking PDAC group. Microorganisms with P < 0.05 and LDA score > 2 were considered significant. F Network analysis of the differential genera between the smoking PDAC group and non-smoking PDAC group. Only the genera with correlation coefficient > 0.5 and P < 0.05 were illustrated in the graph. The red line indicates positive correlation, while the green line indicates negative correlation. The size of the node indicates the abundance of the genus, and the thickness of the lines between nodes indicates the magnitude of the correlation

Fig. 4

Comparison of the metabolome in paracancerous tissue of surgical samples from smoking vs. non-smoking PDAC. A, B, Pancreatic metabolic profile significantly differed between smoking PDAC and non-smoking PDAC group by orthogonal least partial squares discriminant analysis (OPLS-DA) method. Test for the OPLS-DA model showed that the model for this study was valid. C Metabolites with significant differences between groups were presented in the volcano plot. Different metabolites were depicted by setting the fold-change > 1 and P < 0.05 as the threshold. The red points represented the upregulated metabolites (n = 102) and the blue points represented the downregulated metabolites (n = 301). D The altered signaling pathways in the smoking PDAC group compared with the non-smoking PDAC group by KEGG enrichment analysis, marked as *** for P < 0.001, ** for P < 0.01, and * for P < 0.05. E Heatmap representative differential metabolites between the smoking PDAC group and non-smoking PDAC group. The color from blue to red indicates that the expression differential abundance of metabolites is from low to high. The right side shows the VIP bar chart of the metabolite, and the length of the bar represents the contribution value of the metabolite to the difference between the two groups. The color of the bar indicates the significance of the difference between the two groups of samples, P < 0.001 is marked as ***, P < 0.01 is marked as **, P < 0.05 is marked as *

Fig. 5

Correlation analysis between differential bacteria and metabolites of surgical samples from smoking vs. non-smoking PDAC. A tumor tissue; B, paracancerous tissue. The abscissa represents differential bacteria, and the ordinate represents differential metabolites. P < 0.001 is marked as ***, P < 0.01 is marked as **, P < 0.05 is marked as *

Fig. 6

Comparison of the microbiome in FNA samples from smoking vs. non-smoking PDAC. A, B Alpha diversity analysis of the smoking PDAC group (PDAC_smoking, n = 20) and non-smoking PDAC group (PDAC_non-smoking, n = 14). Box plots show the distribution of the Shannon and Sobs indices of alpha diversity scores. C PCoA quantified as weighted-unifrac dissimilarity. D, Distinct bacterial composition at the genus level of subjects between the smoking PDAC group and non-smoking PDAC group. E LDA demonstrated distinct bacterial genera enriched in the smoking PDAC group and non-smoking PDAC group. Microorganisms with P < 0.05 and LDA score > 2 were considered significant. F Network analysis of the differential genera between the smoking PDAC group and non-smoking PDAC group. Only the genera with correlation coefficient > 0.5 and P < 0.05 were illustrated in the graph. The red line indicates positive correlation, while the green line indicates negative correlation. The size of the node indicates the abundance of the genus, and the thickness of the lines between nodes indicates the magnitude of the correlation