Tumor microbiome communities are significantly associated with tumor invasion in resected PTC patients
A total of 80 subjects with PTC were recruited. The demographic and clinical characteristics of PTC were shown in Table 1. Bacterial DNA was extracted from surgically resected PTC tumor and taxonomic profiling via 16S rRNA gene sequencing was performed. The tumor microbial diversity wasused to comparesurgically resected patients who were in differentclinicalstage. The tumor microbial diversity was measured using different methodologies (Sobs,Shannon and Simpson indices), α-diversity of tumor microbiota was significantly lowerin PTC of patients in T1/T2 as compared with that of ones in T3/T4, respectively, calculated byShannon andSimpson index (p=0.0309, p=0.0088 respectively，Wilcoxon rank-sum test; Figure 1 A),which indicated lower microbiota diversity in T1/T2 PTC patients. The microbiota richness was measured by numbers of observed OTUs (Sobsindices), no significant differences were found between different clinical stages. To extend the understanding of the role of microbiome diversity, we aimed to detect whether phylogenetic relationships existed between the bacterial communities enriched in PTC milieu of group in different stages. Beta-diversity were used to generate a principal coordinate analysis (PCoA) using Bray-Curtis metric distances. (Figure 1 C, D). A overlap of OTUs from different groups was revealed, suggesting that the tumor microbial communities exhibited phylogenetic closeness.
Considering the relationship between PTC intra-tumoral bacterial diversity and clinical stages, the general landscape of the tumor microbiome composition was assessed (Figure 2).The enrichment for specific bacterial communities at the phylum taxonomic level was revealed (supplementary Figure S1). At the genus level, Pseudomonas was considered as the clearadvantage bacteria, whereas Rhodococcus, Ralstonia, Acinetobacter and Sphingomonas also present the predominate in PTC intra-tumoral bacterial composition.We next sought to determine if there were differences in the tumor microbiome composition between PTC patients in stage T1, T2, T3 and T4, which revealed the presence of similar communities in PTC patients at different stages (Figure 2 B, C). But obvious difference of the percent of community abundance were also observed (Figure 2 D E).
To further investigate the specific changes of microbiota in tumors of PTC patients of different stages, the relative abundance of taxa was assessed. At the genus level, three genera including Pseudomonas (p=0.0017), Rhodococcus(p=0.02644), Sphingomonas(p=0.0073), displayed different abundance among different stages (Figure 2F and Figure S2 A). Pseudomonasspp the most abundant genus in all the groups, was higher in tumorsof PTC patients in stage T1 and T2, when compared to T3 or T4, respectively(p=0.0049 p=0.0138, p=0.010, p=0.0028, respectively. Figure 2F).Rhodococcuswere also significantly higher in PTC patients in T1 compared to that in T3 (p=0.0032, Figure 2F), and Sphingomonaswere more abudant in T1 and T2compared to T3, respectively (p=0.0001, p=0.0005, respectively. Figure 2F). Then we used these three genera with higher abundance in T1 and T2 to run area under curve (AUC)-receiver operator characteristic (ROC) analysis. The combination of these 3 taxa (Pseudomonas, Rhodococcus, Sphingomonas) resulted in an AUC of 0.82 in the T1_2 and T3_4 (Figure 2 G), while an AUC of 0.9 in the T1 andT4 (supplementary Figure S2 B), which showed the potential ability of the 3-genera microbiome signature to discriminate and influence the PTCtumor invasion status.
The intra-tumor microbial dysbiosis was related to thyroid function
We investigated the effects of the thyroid hormoneson intra-tumor microbiota in PTC patients, which indicated the thyroid function. The association between the thyroid related hormones(FT4, T4， FT3，T3，THS) and microbial abundance were performed by Spearman correlation test. The level of different kind of thyroid hormoneswas related to different microbial genus (Fig 3). Positive relationship was found between FT4 and Neisseria and norank_f__norank_o__Chloroplast, FT3 and Treponema, while negative relationship were showed between FT4 and Klebsiella, T4 and Klebsiella and Escherichia-Shigella, T3 and Granulicatella, TSH and norank_f__norank_o__Clostridia_UCG-014 and Prevotella, respectively. However, all these microbia had relative less richness in PTC intra tumor.
The associationbetweenintra-tumor microbiota and autoimmune thyroid diseases (AITD)-related antibodieswas also investigated, for that the AITD affect the entire metabolism of the human body, and the immunestatus may also contribute to the PTC progression. Autoantibodies against thyroid peroxidase (TPO) and thyroglobulin (TG), charactering the Hashimoto’s thyroiditis (HT), and autoantibodies against thyroid stimulating receptors (TSHR), a marker for Graves’ disease(GD), were involved here.The relationship between microbial abundance and the level of these three autoantibodies, were assessed (Figure 3 B).The data of level of anti-TSHR antibody, anti-TG antibody and anti-TPO was modulated, as that if the data was <0.3 was set as 0.2, <10 was set as 9, <5 was set as 4.Negative correlation between the level of anti-TSHRand Klebsiella and Burkholderia-Caballeronia-Paraburkholderia was found. Positive correlation between the level of anti-TG and Sphingomonas, Rhodococcus, Ralstonia, Brevundimonaswas found, while Anaerococcus and Akkermansia were found to be negatively related to the level of anti-TG. Nine genera including UCG-002, Streptococcus, Parvimonas, Akkermansia, Bacteroides, Haemophilus, Selenomonas, Prevotella and Bifidobacterium showed negative relationship with the level of anti-TPO.
Associations between clinical variables and intra-tumor microbiota
We assessed for potential contributors to microbial diversity, including clinic-pathological features, gender and age. The association of gender bias and intra-tumor microbiome diversity was found in PTC patients. (Fig 4). Higher diversity in the microbiome were observed in females (Fig 4 A), which supposed to have a higher PTC incidence.Of note, Rhodococcus,Ralstonia, Chryseobacterium and Burkholderia-Caballeronia-Paraburkholderia were more abundant in female PTC patients than male patients. (uncorrected p=0.0413, 0.0092 and 0.0275, 0.0008, respectively, Wilcoxon rank-sum test, Fig 4 C). Remarkably, the gender-associated genera were the same PTC T1/T2-enriched genera Rhodococcus. And higher PTC microbiome diversity might show worse association with PTC. However, there was no significant difference in patients at different age or lymphatic metastasis status with respect to α-diversity, β-diversity (supplementary Fig S3).