Characteristics of participants
A total of 50 participants enrolled in the Department of Medical Oncology, The Second Affiliated Hospital of Dalian Medical University (Dalian, China). Participants were excluded after fecal sample collection because of antibiotic use, received chemotherapy, or combined with other diseases such as diabetes mellitus (Fig. 1). Finally, a total of 28 LC patients were remained, they were mainly males (83%) with a median age of 65 years old (Table 1) and with an average smoking index of 606.79±127.1. Average smoking index was the number of cigarettes per day × years of smoking. 19 healthy controls (HC) were included for age and gender matching, with an average smoking index of 350.00±72.1 (P > 0.05 compared with LC group) (details in Table 1). More than 64% of the enrolled LC patients were smokers, with a high smoking index about 606.79±127.1. Among them, 28.57% (8/28) of the patients were diagnosed as SCLC, and 71.43% (20/28) were NSCLC. Among the NSCLC patients, 21.43% (6/28) were diagnosed as squamous cell carcinoma and 50.00% (14/28) were adenocarcinoma.
The overall structure of gut microbiome in LC patients
By sequencing of the 16S ribosomal RNA gene, we found that The HC and LC groups share about 2368 same Operational Taxonomic Units (OTUs), but there are 372 OTUs were obtained specifically in the HC group, and 202 were obtained specifically in the LC group (Fig. 2a). We observed significant decrease in alpha diversity of gut microbiota in LC group, which expressed by the ACE and Chao1 index (Fig. 2b and c). Whereas the Shannon diversity index and the Simpson index did not show significant differences between two groups (Supplementary Fig. S1). Changes in the relative abundance of gut microbes in LC patients were observed not only on the phylum level, but also on the levels of order, class, and family (Supplementary Fig. S2). At genus level, significant decreased abundance of Prevotella and elevated abundance of Bacteroides, and Ruminococcus etc. were detected in LC patients (Fig. 2d).
The beta diversity metrics from the control and LC individuals also showed strong grouping pattern. Although significant inter-individual variation exist among patients and the healthy controls, the fecal microbiota of the two groups still separated clearly according to community composition using Principal component analysis (PCA, Supplementary Fig. S3), and unweighted/weighted UniFrac Principal coordinates analysis (PCoA) (Fig. 2e). These differences were also observed by the two-dimensional Nonmetric Multidimensional Scaling (NMDS) based on unweighted/weighted UniFrac (Fig. 2f). Most of the samples from each group clustered together as evaluated by Hierarchical clustering based on Weighted UniFrac by the method of Unweighted pair-group method with arithmetic means (UPGMA, Supplementary Fig. S4). Especially, when analyzed by the method of Partial Least Squares Discriminant Analysis (PLSDA), we observed a significant separation between the LC patients and HCs (Fig. 2g).
Altered microbiota composition in LC patients
The alteration of gut microbiota in LC patients was further proved by the LEfSe approach, which identified the key phylotypes responsible for the difference between the two groups. Actinobacteria, Bacilli, Ruminococcus, Streptococcus, and Mycobacteriaceae, etc, which were most abundant in the LC group, and Prevotella, Bacteroidetes, and Dialister, which were most abundant in the HCs, were the dominant phylotypes that contributed to the difference between the intestinal microbiota of LC patients and HCs (Fig. 3a and b). The significantly elevated relative abundance of Actinomyceae, Streptococcus, and Ruminococcus, and decreased abundance of Prevotellaceae were observed in the gut microbiome of most of the LC patients, which suggested a highly consistence among different individuals (Supplementary Fig. S5).
Using the method of Metastats, we found a significant decrease in the abundance of Bacteroidetes (phylum), Bacteroidia (class), Bacteroidales (order) and elevated abundance of Firmicutes (phylum), Bacilli (class), Actinomycetales (order), Bacillales (order), Lactobacillales (order) in gut of the LC patients (Supplementary Fig. S6). On the family level, significant elevation of the relative abundance of Streptococcaceae, Actinomycetaceae, decreased abundance of the Prevotellaceae and Veillomellaceae were observed in gut of LC patients compared with HCs (Fig. 4a). These changes may mainly due to the changing of genus such as the Streptococcus, Actimomyces, and Prevotella etc (Fig. 4b). In addition, we also detected elevation of genera such as Ruminococcus, Rothia, Bacillus, Peptostreptococcus, Mycoacterium, etc, and decreased abundance of Dialister in gut of LC patients, which are consistent with LEfSe analysis results.
Comparison of gut microbiome in LC patients with specific histological types
We further performed a detailed comparison of the gut microbiome in lung cancer patients according to different histological types, including adenocarcinoma (Group A, n=14), squamous cell carcinoma (Group B, n=6), and SCLC (Group C, n=8). Only the index of alpha diversity (ACE) in SCLC patients is significantly lower than control (Fig. 5a). Other groups showed no significant differences compared either with HC or other groups, although the average indexes of each of the specific histological type are obviously lower than that of the HCs (Fig. 5a and Supplementary Fig. S7). The beta diversity analysis by PCoA and NMDS showed no obvious separation between groups (Fig. 5b and c). When analyzed by the method of PLSDA, we observed a separation between group A and C, which suggested that the gut microbiome of SCLC patients may differ from that of LC patients with adenocarcinoma (Fig. 5c). The taxonomy-based comparison at the genus level showed that, Bifidobacterium, Clostridium, and Prevotella, etc, are the dominant phylotypes in group A, but were significantly reduced in the other two groups. In group B, the dominant genera including Ruminococcus, Lachnospira, and Lactobacillus, etc, which are less abundant in group A and C. in addition, the genera of Streptococcus, Anaerotruncus, and Bacillus, etc are more abundant in group C when compared with group A and B (Supplementary Fig. S8).