Lung microbiome alterations in NSCLC patients

Abstract

Lung is colonized by a diverse array of microbes and the lung microbiota is profoundly involved in the development of respiratory diseases. There is little knowledge about the role of lung microbiota dysbiosis in lung cancer. In this study, we performed metagenomic sequencing on bronchoalveolar lavage (BAL) from two different sampling methods in non-small cell lung cancer (NSCLC) patients and non-cancer controls. We found the obvious variation between bronchoscopy samples and lobectomy samples. Oral taxa can be found in both bronchoscopy and lobectomy samples and higher abundance of oral taxa can be found in bronchoscopy samples. Although the NSCLC patients had similar microbial communities with non-cancer controls, rare species such as Lactobacillus rossiae, Bacteroides pyogenes, Paenibacillus odorifer, Pseudomonas entomophila, Magnetospirillum gryphiswaldense, fungus Chaetomium globosum et al. showed obvious difference between NSCLC patients and non-cancer controls. Age-, gender-, and smoking-specific species and EGFR expression-related species in NSCLC patients were detected. There results implicated that different lung segments have differential lung microbiome composition. The oral taxa are found in the lobectomy samples suggesting that oral microbiota are the true members of lung microbiota, rather than contamination during bronchoscopy. Lung cancer does not obviously alter the global microbial composition, while rare species are altered more than common species. Certain microbes may be associated with lung cancer progression.

Figure 1

Overall microbiome composition of lungs. (A) Taxonomic composition of non-human sequences at species level across 47 samples. (B) Distance based redundancy analysis (RDA) with 4 explanatory variables age, sampling methods, disease and smoking history affecting the microbial community composition. Variables were plotted as points. NMDS analysis of morphological variation of 47 samples collected by 2 sampling methods in the NSCLC patients and non-cancer controls were shown in (C) and (D). Colorful dots indicate different sampling methods or disease cases. Dotted lines display ellipses, which represent the 95% confidence interval. Anosim was performed to test statistically whether there is a significant difference. p < 0.05 was considered to be significant. B bronchoscopy samples, L lobectomy samples, N non-cancer controls, T NSCLC patients.

Figure 2

The microbial composition varied in different anatomy sites. (A) The schematic diagram of the sampling sites. The squared area represents the extent of bronchoscopy sampling area. The circled area covers the sampling area from lobectomy. (B) The Shannon diversity index of bronchoscopy samples and lobectomy samples. Difference between sampling methods was determined by Student t test. ***p < 0.001. (C) Taxonomic composition at phylum level in bronchoscopy and lobectomy samples. Neutral model applied to compare the overall lung microbiota composition at genus level (D) and at species level (E). Dashed lines represent 95% confidence intervals around the model prediction (solid line). The green dots that are beyond the 95% confidence intervals are the OTUs whose observed frequency in lobectomy samples is greater than the model prediction (bronchoscopy samples). The dark golden points that are beyond the 95% confidence intervals are the OTUs whose observed frequency in lobectomy samples is less than the model prediction (bronchoscopy samples). (F)Taxonomic composition of lobectomy samples at genus level in NSCLC patients and non-cancer controls. The differential genes between NSCLC patients and non-cancer controls in lobectomy samples were identified by Wilcoxon rank-sum test (p < 0.05). (G) Taxonomic composition of lobectomy samples at genus level in NSCLC patients and non-cancer controls. The differential genes between NSCLC patients and non-cancer controls in lobectomy samples were identified by Wilcoxon rank-sum test (p < 0.05). B bronchoscopy samples, L lobectomy samples.

Figure 3

Microbiome composition of NSCLC patients and non-cancer controls in bronchoscopy samples. (A) Taxonomic composition of non-human sequences at phylum level in NSCLC patients and non-cancer controls in bronchoscopy samples. (B) The Shannon diversity index between NSCLC patients and non-cancer controls in bronchoscopy samples. Difference between sampling methods was determined by Student t test. *p < 0.05. (C) PCoA analysis of NSCLC patients and non-cancer controls in bronchoscopy samples. (D) The differential species between NSCLC patients and non-cancer controls in bronchoscopy samples were identified by Wilcoxon rank-sum test (p < 0.05). N non-cancer controls, T NSCLC patients.

Figure 4

Microbiota composition of NSCLC patients and non-cancer controls in lobectomy samples. (A) Taxonomic composition of non-human sequences at phylum level in NSCLC patients and non-cancer controls in lobectomy samples. (B) The Shannon diversity index between NSCLC patients and non-cancer controls in lobectomy samples. Difference between sampling methods was determined by Student t test. NS no significance. (C) PCoA analysis of NSCLC patients and non-cancer controls in lobectomy samples. Anosim was performed to test statistically whether there is a significant difference. p < 0.05 was considered to be significant. (D) The differential species between NSCLC patients and non-cancer controls in lobectomy samples were identified by Wilcoxon rank-sum test (p < 0.05). N non-cancer controls, T NSCLC patients.

Figure 5

EGFR expression related microbiota composition in NSCLC patients. Representative images of the immunohistochemical detection of EGFR (A). Magnification: ×100. (B) The differential species in bronchoscopy samples between EGFR high patients and EGFR low patients were identified by Wilcoxon rank-sum test (p < 0.05).