- The structural composition of saliva microbiota was altered in patients with high TROP2 expression and
Saliva samples from 11 subjects, including 5 patients diagnosed with OSCC and 6 volunteer subjects, were collected from February to April 2022 from Nanjing Stomatological Hospital. The mean age of the total cohort was 56.4 years and comprised 50% men. Based on the diagnostic results, TROP2 expression in oral tissues of OSCC patients and volunteers was detected by immunohistochemistry staining, and the expression was 56.70% in OSCC tissues, which was higher than that in normal oral tissues (16.45%) (Fig. 1 G,H).
To investigate the characteristics of the saliva microbiome, we analyzed the bacterial composition and community structure via 16S rDNA gene sequencing. Based on the microbial genomics samples, an average of 806,197 reads were aligned per sample. Alpha diversity based on Chao1 distance and beta diversity based on an unweighted principal coordinate analysis (PCoA) showed that the bacterial diversity of OSCC indicated an ecological resemblance among samples in each group, revealing a marked decrease and distinguishing it from the HC group (Fig.1 A,B), respectively. Although microbial variability at the phylum level cannot explain individual differences, we can roughly distinguish the difference between the two groups from the microbiome relative abundance. The dominant bacterial species of each group were similar and belonged to five main phyla: Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, and Fusobacteria. Moreover, levels of Elusimiceobia, Chloroflexi, Verrucomicrobia, Deferribacteres, and Planctomycetes increased in the OSCC group, while levels of Synergistetes, Candidatus-Gracilibacteria and Cyanobacteria decreased slightly (Fig. 1C). We further analyzed the taxonomic profile at the genus level, and the relative abundance among the 30 major genera showed that Prevotella, Treponema, Lautropia, and Lactobacillus were markedly decreased (Wilcox. test. p<0.05) in OSCC patients, while Aggregatibacter and Rothia were relatively increased (wilcox. test. p<0.05) (Fig.1D). Next, we compared wilcox. test. P values for the top 30 species of bacteria between the OSCC group and HC group at the species level. Five species, including Lactobacillus casei, Neisseria subflava, uncultured Haemophilus sp., Veillonella sp. HGFM67 and Veillonella sp. oral clone VeillB9 were significantly decreased (Wilcox. test.p<0.05) while Veillonella parvula was relatively decreased((wilcox. test.p=0.05)) in OSCC patients. We also found that two species, Bacilli unclassified and Rothia mucilaginosa, were significantly increased in the OSCC group (Fig.1 E). We further analyzed the bacterial community structure by using linear discriminant effect size (LEfSe), an algorithm for bacterial biomarker discovery that uses linear discriminant analysis (LDA) to estimate the effect size of each taxon that is differently represented in two groups, and found several high-dimensional biomarkers in two groups (Fig. 1F ). These results indicated that the composition of the saliva microbiota in patients with high TROP2 expression was significantly altered in OSCC patients.
- The screening and identification of Veillonella parvula NCTC11810
Veillonella species are known as early colonizers of the oral microbiome and normally inhabit the mouth, upper respiratory tract, intestine and vagina[31, 32]. Recent studies illustrate the association of Veillonella spp. with mixed infections in children. However, the function and importance of Veillonella species in OSCC cancer are uncertain[33].
Since Veillonella sp. HGFM67, Veillonella sp. oral clone VeillB9 and Veillonella parvula were decreased in OSCC patients to different degrees. To investigate the difference in Veillonella between the two groups in vivo, we collected 3 more samples of control group and analyzed the colony forming units (CFUs) of the Veillonella genus on Veillonella agar medium and found that the CFUs of Veillonella bacteria were not different in OSCC patients compared with those in the HC controls, which was consistent with the taxonomy profile results (Fig. 2 A,B and Fig. 1,D). For further exploration, we purified 34 colons from columbia blood plates by dilution separation methods from HC saliva samples and identified one colon of Veillonella parvula NCTC11810 with 99.71% sequence similarity (Supplementary Fig.S1). Next, we detected the expression level of Veillonella parvula NCTC11810 by real-time PCR and found a 10-fold reduction in the OSCC group (Fig. 2C).
- Veillonella Parvula NCTC11810 Cells and Culture SupernatantInhibited the Proliferation and Invasion Ability of HN6 Cells
As in our previous study, the expression of TROP2 was evaluated in HN6 cells compared with HOK cells (Fig.3A). To investigate the biological function of Veillonella parvula NCTC11810 in OSCC cells, we treated HOK and HN6 cells with Veillonella parvula NCTC11810 cells, heat inactivated Veillonella parvula NCTC11810 cells and culture supernatant for 2 hours. The viability of cells was investigated by CCK8 assays, and the results showed that cell proliferation at 24 hours was significantly reduced in HN6 cells treated with Veillonella parvula NCTC11810 and culture supernatant fluid compared with heat inactivated bacterial cells. For another cell line, Veillonella parvula NCTC11810, heat inactivated Veillonella parvula NCTC11810, and the culture supernatant treatment seemed to have no effects on the proliferation ability of HOK cells (Fig.3 B). In transwell assays, the number of cells that migrated across the Matrigel was counted, and the results indicated that both culture supernatant and bacterial cells reduced the cell invasion ability 24 hours after treatment in HN6 cells, but heat inactivated Veillonella parvula NCTC11810 cells could not inhibit the invasion of HN6 cells; however, the number of HOK cells that migrated across the Matrigel was not different among the treatment groups. (Fig.3C,D)
The data above indicated that the viable Veillonella parvula NCTC11810 cells and its metabolite products inhibited the proliferation and invasion ability of HN6 cells.
- Veillonella Parvula NCTC11810 Cells and Culture Supernatant Promoted the Apoptosis of HN6 Cells
To further confirm the function of Veillonella parvula NCTC11810 in OSCC cells. HOK and HN6 cells were treated with the measures above, subjected to PI/Annexin V staining and analyzed by flow cytometry analysis to determine the percentages of viable cells (lower left quadrant), early apoptotic cells (lower right quadrant), and late apoptotic cells (upper right quadrant). The results revealed that HN6 cells in the late apoptotic stage accounted for 9.25% in the Veillonella parvula NCTC11810-treated group and 17.48% in the culture supernatant-treated group, which were markedly increased compared with those of the heat inactivated Veillonella parvula NCTC11810-treated and control groups. The percentage of late apoptotic HOK cells was not different among the Veillonella parvula NCTC11810, heat-inactivated Veillonella parvula NCTC11810 and culture supernatant groups compared with those of the control group. (Figure 4 )
The data above indicated that apoptosis was significantly induced by the viable Veillonella parvula NCTC11810 cells and its metabolite products in HN6 cells.
The cytological phenotype of the Veillonella parvula NCTC11810 cell treatment but not the heat-inactivated Veillonella parvula NCTC11810 cell treatment inhibited the proliferation and invasion and promoted the apoptosis of HN6 cells might be because Veillonella parvula NCTC11810 cells were added to the cell culture plates, and the nutrient substance in the culture medium might promote the metabolism of Veillonella parvula NCTC11810 cells to some degree and induce the secretion of SCFAs, which play an antitumor function in HN6 cells.
The results suggested that the metabolite products of Veillonella parvula reduced proliferation and invasion and promoted apoptosis in TROP2-overexpressing HN6 cells. In the human oral cavity, the enriched culture of anaerobic microorganisms, for example Veillonella parvula, was shown to favor the process of fermenting lactic acid into propionate through the acrylate pathway[34-36]. Propionate has been found to inhibit the proliferation of liver cancer cells and induce the apoptosis of breast cancer cells, indicating a potential anticancer property; however, studies on propionate for the proliferation, invasion and apoptosis of OSCC are lacking. Thus, we investigated the anticancer effect of sodium propionate (SP) on the proliferation, invasion and apoptosis of OSCC cells.
- SP inhibited the proliferation and invasion of HN6 cells of OSCC.
To determine the regulatory mechanism of Veillonella parvula in OSCC cells with high TROP2 expression, we treated HN6 and HOK cells with sodium propionate (SP) in diluted concentrations of 10 mM, 20 mM, 50 Mm and 100 mM. Proliferation was investigated by CCK8 assays 24 hours later, and the data suggested that cell viability was affected by the concentration of SP, as a stronger inhibition ability appeared in HN6 and HOK cells treated with higher concentrations of SP. Furthermore, the inhibition ability of SP was more distinct in HN6 and HOK cells at concentrations of 10 mM and 20 mM (Fig. 5A). Then, we detected the invasion ability of HN6 cells. The results of the transwell assay 24 hours after 10 mM SP treatment indicated that the invasion ability of HN6 cells was significantly decreased in the SP group, while there was no obvious change in HOK cells (Fig.5B,C). Even the low concentration of SP that our data showed may inhibit the proliferation and invasion ability of OSCC cells with high TROP2 expression compared with that of normal cells.
- Exposure to SP induced apoptosis in HN6 cells.
Since the culture supernatant of Veillonella parvula NCTC11810 promoted the apoptosis of HN6 cells, cells can take up propionate and use it as a metabolic substrate, providing energy through mitochondrial oxidation[37]. To further investigate the biological function of propionate in HN6 cells, 10 mM SP was added to HN6 cells for 24 hours, stained with PI/Annexin V and analyzed by flow cytometry . The results demonstrated that the overall apoptosis rate of SP-treated HN6 cells was 33% and significantly increased compared with that of the HOK group (3.36%); in particular, the late apoptotic percentage was 25.56% in HN6 cells, and the early apoptotic percentage was 7.97%, which were higher than those of HOK (3.37% and 0.25%, respectively) (Fig.6 and Supplementary Fig.S2).
- SP Inhibited the TROP2/PI3K/Akt Pathway in HN6 Cells
Our previous study indicated that TROP2 might induce cell proliferation and invasion by activating the PI3K/Akt signaling pathway in OSCC, osteosarcoma and gallbladder cancer cells[38, 39]. To detect whether SP regulates cell behavior by modulating the Trop2/PI3K/Akt pathway, Western blotting was performed to explore the expression of related proteins in HN6 and HOK cells. As shown in Fig.7, the expression levels of PI3K and Akt were not significantly changed between the SP treatment and control groups in HOK and HN6 cells (Fig. 7A,B). However, the expression levels of phosphorylated PI3K and phosphorylated Akt were totally inhibited in HOK cells and SP-treated HOK and HN6 cells but relatively activated in HN6 cells (Fig. 7C,D). Moreover, the expression level of TROP2 in SP-treated HN6 cells was remarkably inhibited compared with that in HN6 control group(Figure.7E).
These results suggest that SP, the main metabolite of Veillonella parvula NCTC11810, may inhibit proliferation and invasion ability and promote the apoptosis by affecting the activity of the TROP2-related PI3K/Akt pathway.