Aspirin prevents colorectal cancer by regulating the abundance of Enterococcus cecorum and TIGIT+Treg cells

Abstract

Although aspirin can reduce the incidence of colorectal cancer (CRC), there is still uncertainty about its significance as a treatment for CRC, and the mechanism of aspirin in CRC is not well understood. In this study, we used aspirin to prevent AOM/DSS-induced CRC in mice, and the anti-CRC efficacy of aspirin was assessed using haematoxylin and eosin (H&E) staining and by determining the mouse survival rate and tumour size. 16S rDNA sequencing, flow cytometry (FCM), and Western blotting were also conducted to investigate the changes in the gut microbiota, tumour immune microenvironment, and apoptotic proteins, respectively. The results demonstrated that aspirin significantly exerted anti-CRC effects in mice. According to 16S rDNA sequencing, aspirin regulated the composition of the gut microbiota and dramatically reduced the abundance of Enterococcus cecorum. FCM demonstrated that there were more CD155 tumour cells and CD4 + CD25 + Treg cells showed increased TIGIT levels. Moreover, increased TIGIT expression on Treg cells is associated with reduced Treg cell functionality. Importantly, the inhibition of Treg cells is accompanied by the promotion of CD19 + GL-7 + B cells, CD8 + T cells, CD4 + CCR4 + Th2 cells, and CD4 + CCR6 + Th17 cells. Overall, aspirin prevents colorectal cancer by regulating the abundance of Enterococcus cecorum and TIGIT + Treg cells.

Figure 1

Antitumour effects of aspirin in a CRC mouse model. (a) Haematoxylin and eosin staining of colorectal tissue from mice. Scale bar: 50 um. n = 3. (b) Survival rate of CRC mice. (c) Comparison of the tumour volume. n = 3. (d) Body weight of mice during the experiment. (e) Protein was extracted from mouse tumour tissue for Western blotting to determined BAX expression (original blots/gels are presented in Supplementary Fig. S2). n = 4. *p < 0.05, **p < 0.01, ***p < 0.001 compared to RC.

Figure 2

Aspirin altered the gut microbial composition in the CRC mouse model. (a) Rank abundance and species accumulation boxplot. The rank abundance was obtained by plotting the ranking number of OTUs as the horizontal coordinate and the relative abundance as the vertical coordinate. A species accumulation boxplot was generated for the biodiversity and community survey. (b) Venn diagram at weeks 4, 7, 10, and 13. A Venn diagram was generated based on the OTU analysis. (c) Nontetric multidimensional scaling at weeks 4, 7, 10, and 13. NMDS is a nonlinear model based on the Bray‒Curtis distance for analysis.

Figure 3

Aspirin reduces the abundance of Enterococcus cecorum in a CRC mouse model. (a) Relative abundance histogram at the genus levels. To determine the relative abundances, the number of tags corresponding to a species in a sample at a certain classification level was divided by the total number of tags corresponding to the OTUs clustered in the sample. (b) MetaStat diagrams of the species levels at week 13. *p < 0.05, **p < 0.01, ***p < 0.001 compared to RC.

Figure 4

Aspirin decreases CD4 + CD25 + Treg cells and activates CD19 + GL-7 + B cells, CD8 + T cells, CD4 + CCR4 + Th2 cells, and CD4 + CCR6 + Th17 cells. Representative dot plots showing the flow cytometry gating strategy used for CD11b + F4/80 + Møs and CD161 + NK cells (a), CD4 + CD25 + Treg cells (b), CD19 + GL-7 + B cells, Perforin + CD8 + T cells, granzyme B + CD8 + T cells, CD4 + CXCR3 + Th1 cells, CD4 + CCR4 + Th2 cells, CD4 + CXCR5 + Tfh cells and CD4 + CCR6 + Th17 cells (c–e) in the mouse spleen. *p < 0.05, **p < 0.01, ***p < 0.001 compared to RC.

Figure 5

Aspirin downregulates the expression of TIGIT on Treg cells. (a,b) Representative dot plots showing the flow cytometry gating strategy used for CD155-expressing tumour tissue and adjacent tissue samples and for TIGIT-expressing T cells. (c) Correlation analysis between the expression of TIGIT on Treg cells and the abundance of Enterococcus cecorum, Clostridium disporicum, and Escherichia coli. *p < 0.05, **p < 0.01, ***p < 0.001 compared to RC.

Figure 6

Schematic diagram of the function of aspirin in CRC. Aspirin can prevent colorectal cancer by regulating the abundance of Enterococcus cecorum and TIGIT + Treg cells.