Oral microbiota in EsoSCC
To characterize the oral microbiome community in EsoSCC, oral biofilms over dental surfaces were collected from EsoSCC patients and controls. As shown in Suppl. Figure 1a-b, ESCC samples differed from controls in oral microbiome α-diversity, (species richness) and β-diversity (microbiome composition). There was a significant difference in the overall oral microbiome composition between the cancer and control groups (p=0.001), as measured by Adonis analysis. To identify the differentially enriched species within groups, the LEfSe method was used. Streptococcus mutans, Veillonella parvula, Streptococcus peroris, and P. gingivalis (PG) were abundant in the oral biofilms from EsoSCC samples (Figure 1a). To further examine if PG is more frequent in esophageal cancer tissue, we employed qPCR to examine the presence of PG 16S rDNA in fresh esophageal specimens. The data revealed that PG16S rDNA were more frequent detected in EsoSCC specimen than non-malignant specimen (Figure 1b).
Positive PG staining correlates with the prognosis of EsoSCC
Figure 1c shows representative positive and negative stained slides for PG in human EsoSCC tissue specimens at diagnosis. The IHC data for TMA slides from 21 EsoSCC specimens revealed that PG was detected more frequently in 12 (57%) cancerous tissues than in adjacent esophageal mucosa (24%). Since an association between PG infection and EsoSCC was noted, we examined whether positive staining with anti-PG antibodies was associated with the prognosis of EsoSCC. The clinical characteristics of the EsoSCC patients are presented in Table 1. For the total 156 EsoSCC specimens, 89 (57%) showed the presence of PG. Positive staining for PG was positively related to poor differentiation and advanced stage in EsoSCC patients (p < 0.05). Moreover, the presence of PG was significantly associated with a higher disease failure rate (developing locoregional recurrence and distant metastasis) and reduced overall survival rate (Figure1d & Suppl. Fig 2). These findings suggested that the presence of PG contributes to tumor aggressiveness and poor prognosis in EsoSCC.
PG infection-associated esophageal tumorigenesis in 4NQO-induced tumor model
We established and characterized an experimental mouse model featuring 4NQO-induced esophageal cancer and PG infection, as described in the Methods. At 12~14 weeks after the 16-week 4NQO treatment, the FMT images were taken, and then the mice were analyzed, and the esophagus was removed for further evaluation. Figure 2a demonstrated the lesions on the esophagus in 4NQO-treated mice, and the pathological esophageal lesions including hyperplasia/papilloma, carcinoma in situ (CIS), and invasive carcinoma. The validity of FMT at 12 weeks after the 16-week 4NQO treatment for detecting esophageal lesions in mice is shown in Figure 2b. Furthermore, esophageal carcinoma (CA) including carcinoma in situ (CIS) and invasive carcinoma, had a significantly higher glucose-uptake signal than those with benign tumor (hyperplasia and papilloma). We also examined the link between tumor progression and MDSC recruitment in mice. There were significantly higher MDSCs in mice with CA (Figure 2c). In addition, PG infection was associated with the increases in glucose-uptake signal by FMT and MDSC recruitment by FACS in 4NQO-treated mice (Figure 2d-e).
PG enhanced tumor invasion
To examine whether the invasiveness of cancer cells was modulated by PG, we performed an in vitro invasion assay to analyze the invasiveness of PG-infected EsoSCC cells. Figure 3a shows that CE81T and TE2 cells coincubated with PG had a prominent increase in invasive potential compared with control cells. Epithelial-mesenchymal transition (EMT) play a critical role in the invasiveness of cancer , and we examined whether it is the underlying mechanism responsible for the enhanced tumor aggressiveness induced by PG infection. As shown in Figure 3b, PG-infected EsoSCC cells had augmented EMT changes and presented with increased β-catenin and matrix metalloproteinase (MMP)-9 expression and decreased E-cadherin expression. In addition, cancer stem cells (CSCs) were reported to be key event in aggressive tumor behavior , including upper aerodigestive tract cancer. As shown in Figure 3c-d & Suppl. Figure 3, coincubation with PG significantly stimulated the expression of CD44 and ALDH1 which are CSC markers in human esophageal cancer cells.
The correlation between PG infection and IL-6 signaling
We previously reported that overexpressed IL-6 correlated with the poor prognosis of EsoSCC, and activation of the IL-6/STAT3 pathway plays a critical role in CSC formation and EMT [20, 21]. Furthermore, activated IL-6/STAT3 signaling has been shown to be the adaptive pathway contributing to microbiota-induced tumor progression . Accordingly, we investigated the correlation between PG and IL-6 signaling. Figure 4a-b shows that PG significantly increased IL-6 expression in cancer cells and the supernatant in cell cultures. Furthermore, a significantly positive correlation was found between cancer specimens that expressed IL-6 and the presence of PG (Fig. 4c & Table 1). It has been reported that bacterial infection-related autophagy may have an effect on tumor initiation and cancer treatment [3, 22]. PG is reported to activate cellular autophagy and stimulate pro-inflammatory cytokines . As shown in Figure 4d-e & Suppl. Figure 4, the PG-infected cells had increased autophagy, represented by the conversion of LC3-I to LC3-II, associated with increased IL-6 expression. Furthermore, pre-treatment with the autophagy inhibitor 3-MA 10 mM for 1 h attenuated the expression of IL-6 in PG-infected cells. The results reveal that autophagy may be one of the mechanisms contributing to the increased IL-6 noted in PG-infected cancer cells.
Effect of calcitriol on the invasiveness of tumors with PG infection
In the gut, calcitriol was reported to have anti-inflammatory and anti-infectious effects in experimental models . We previously reported that calcitriol abrogated IL-6-induced tumor aggressiveness and inhibit the promotion of esophageal tumor. Accordingly, we examined whether calcitriol attenuates the tumor-promoting status induced by PG infection. As demonstrated in Figure 5a, calcitriol inhibited the PG-induced increase in IL-6 and activated STAT3 in esophageal cancer cells. In vitro, incubation with calcitriol for 48 h attenuated the invasion ability in association with reversing the expressions of β-catenin, MMP-9 and ALDH1 induced by PG infection (Figure 5b-d & Suppl. Figure 5). Based on the data, we suggest that calcitriol reverses EMT and CSC changes and subsequently attenuates tumor invasiveness in PG-infected esophageal cancer, which is mediated by regulating IL-6 signaling, at least a part.
Increased calcitriol induction by UVB light reverses the tumor-promoting status induced by PG infection
The concentrations of calcitriol required for antineoplastic effects are usually too high to achievable safely in patients with vitamin D3 is dosed daily. Therefore, we examined the effect of increased calcitriol induced by UVB irradiation with a 300 nm LED. As shown in Figure 6a, after UVB irradiation, serum calcitriol increased markedly in mice. To investigate whether UVB light treatment ameliorates the induction of esophageal cancer, mice were irradiated per a chronic exposure protocol. As shown in Figure 6b & Suppl. Figure 6a-b, UVB light treatment increased calcitriol in serum, with decreased IL-6 in serum, attenuated MDSC recruitment and a lower incidence of esophageal CA formation. Moreover, an orthotopic tumor implantation model was used to examine the effect of UVB light treatment on xenograft tumor growth. As demonstrated in Figure 6c-e, the UVB light treatment decreased the glucose-uptake signals, decreased tumor size and attenuated EMT change.