OBJECTIVE  To evaluate the tumor suppressive effect of Huaier n-butanol extract in gastric cancer-bearing mice and its effect on gut microbiome, and to explore the role of specific flora in gastric cancer as a biomarker.

METHODS  Mouse forestomach carcinoma(MFC) tumor-bearing mouse model was constructed, Huaier n-butanol extract was administered daily, and the body weight and tumor diameter were monitored daily. Additionally, the volume and weight of the tumor mass were measured after the completion of the experiment. Immunohistochemical staining experiments were performed on mouse tumors to detect the expression of Ki67, Bcl-2, CD3, CD4, CD8, CD11c, FOXP3, and RORγt, and to evaluate the proliferation, apoptosis and immune activity of tumor cells in the tumor mass. Furthermore, 16S rRNA high-throughput sequencing of mouse feces was performed to explore the effect of the extract on the structural composition and diversity of the gut flora. Finally, qPCR experiments targeted the specific and differential gut microbiome in gastric cancer tissues to explore the correlation between their abundance and prognosis in patients with advanced gastric cancer.

RESULTS  Huaier n-butanol extract can inhibit the growth of gastric cancer in mice. The extract can significantly promote CD3, CD4, CD11c, RORγt expression, and inhibit FOXP3 expression, affecting the tumor immune microenvironment. In the mouse subcutaneous tumor model, pre-use of antibiotics destroyed the gut microbiome, which then affected the immune microenvironment in the tumor and the anti-gastric cancer effect of Huaier n-butanol extract. After the intervention of Huaier n-butanol extract, the abundance of Firmicutes in the intestinal flora of mice was relatively reduced, while the abundance of Proteobacteria and Bacteroidetes were relatively increased, and bacterial clusters were separated. From the level of Genus, Rikenellaceae_Rc9_gut_group, Duncaniella, Oscillibacter, Eubacterium nodatum group have relatively increased, and Roseburia, Anaerotruncus, and Rikenella have relatively decreased, suggesting that the intestinal flora have changed in the process of cancer inhibition by Huaier n-butanol extract.

CONCLUSION This study showed that the Huaier n-butanol extract could inhibit the tumor growth and affect the tumor immune microenvironment of MFC tumor-bearing mice. In addition, Huaier n-butanol extract affected the richness and diversity of gut microbiome in tumor-bearing mice during the process of tumor inhibition. Moreover, the selected specific differential bacteria provide a new idea for predicting the prognosis of gastric cancer patients.