| Cancer is the leading cause of death in the world.In 2020,there were 19.3 million new cancer cases and 10 million deaths worldwide.Breast cancer and colorectal cancer were the most common cancers with incidence rates of 11.7% and 10%,respectively.Therefore,performing innovation of tumor treatment methods and individualized tumor treatment based on tumor biology is of great significance for the precise prevention and control of tumors.Radiotherapy,as one of the main methods of treatment,accounts for about 70% of all tumor treatments.The side effects of conventional radiotherapy,such as higher radiation tolerance and lower sensitivity,limit its application in precision treatment of cancer.Currently,carbon ion beam(12C6+)has become the most advanced radiotherapy beam due to its unique depth dose distribution,higher relative biological effect(RBE),and lower damage to normal tissues,especially for the treatment of refractory tumors that are inoperable and insensitive to conventional radiation.Gut microbiota is closely related to the efficacy,prognosis effects of radiotherapy.Radiotherapy can result in dysbiosis of gut microbiota,destruction of intestinal mucosa and changes in intestinal permeability,which leads to enterotoxins entering the blood circulation and endangering the health of the host.However,the damage to gut microbiota caused by radiotherapy and its harm have not been extensively paid attention to by the medical community,especially there is a lack of effective solutions.Probiotics are recognized as safe and effective modulators of intestinal microbiota,which show promising prospects in reducing the injury to gut microbiota caused by radiotherapy and improving efficiency of cancer treatment.In addition,the mechanisms by which probiotics regulate gut microbiota to improve the efficacy of radiotherapy remain largely unknown.Therefore,in this study,the anti-tumor probiotics were employed to evaluate their effect and underlying mechanism in prevention of colon cancer and adjuvant carbon ion radiotherapy for tumor models in mice.Through multiomics analysis of intestinal microbiome,metabolome and tumor transcriptome,we explored the mechanism by which probiotics improve the therapy outcomes and identified the molecular targets for cancer prevention and treatment,which will provide an important theoretical foundation for the establishment of tumor precision treatment programs based on microbial therapy and the development of anti-tumor drugs.The main results of this paper are as follows:(1)The study on breeding of anti-tumor probiotics and its colonization in the intestines of mice.Lactobacillus reuteri JMR-01 was isolated from the intestinal tract of mice with a JY300-8 adjuvant carbon ion-cured breast cancer model.In vitro,JMR-01 combined with JY300-8 inhibited the proliferation of Tubo cell by 86.74%,which is higher than that reported in similar studies.Based on q PCR technology,the gut microbial targeted primer(Tot)was employed as the internal reference,and a 16 S targeted primer that designed for probiotic was chosen as the specific amplification primer to establish a noninvasive colonization detection system for probiotics in mice.After 6 days of continuous gavage of probiotics(JY300-8 and JMR-01),the relative abundance of JY300-8 and JMR-01 in the intestinal tract of mice was 7.085 and 6.159 times higher than that in the control group,respectively,indicating that the probiotics has been successfully colonized in intestine of mice.In addition,continuous and highdose gavage of probiotics(JY300-8 and JMR-01)showed no toxic side effects in mice,which could be safe and further study on the prevention and treatment of tumor in preclinical animal model.These results provide a new strain resource and experimental basis for the prevention and treatment of cancer with probiotics.(2)Evaluation of the therapeutic effect of probiotics on HER2-positive breast cancer models in mice.Although alone probiotics(JY300-8 and JMR-01)could not significantly inhibit the proliferation of breast cancer cells in mice,orally administering JY300-8 and JMR-01 to tumor-bearing mice prolonged their overall survival rate.The survival rate of tumor-bearing mice in the tumor control group was 0,while that in the live probiotics treatment group was 35%,which was significantly higher than that in the control group(0)and the inactivated bacteria group(10%)(p < 0.05).In addition,probiotics can balance dysbiosis of intestinal flora induced by tumor and restore them to a healthy level.The result suggests that probiotics(Y300-8 and JMR-01)have the potential to enhance the therapeutic effect of cancer by regulating intestinal flora,which provides a good experimental foundation and feasibility guarantee for further improving the treatment efficiency of breast cancer.(3)The study on probiotics adjuvant carbon ion radiotherapy improves the therapeutic efficacy of HER2 positive breast cancer models in mice.To further improve therapeutic efficacy of breast cancer models in mice,mice with subcutaneously transplanted tumors expressing high levels of the HER2 gene were selected as research subjects to investigate the effect of carbon ion radiotherapy by intervention of probiotic.The study showed that carbon ion radiotherapy increased the survival rate and complete remission rate of breast cancer model in mice(202 d)by 38.46% and 23.08% compared with the tumor control group,respectively.The carbon ion radiotherapy reduced the risk of lung metastasis in tumor-bearing mice by 33%.Moreover,probiotics adjuvant carbon ion radiotherapy further improved the survival rate and complete remission rate of breast cancer models in mice(202 d),which were 61.54% and 41.23% higher than those of the tumor control group,respectively.Particularly,no lung metastases were observed in tumor-bearing mice treated with probiotics adjuvant carbon ion radiotherapy.HE staining of the small intestine revealed that radiation caused an increase of plasma cells and the accumulation of lymphocytes in the basal part of the intestine,and the destruction of the integrity of the intestinal structure in breast cancer models in mice.Probiotics adjuvant carbon ion radiotherapy balances the intestinal flora dysbiosis induced by radiotherapy and reduces small intestinal inflammation and injury in breast cancer models in mice.In particular,probiotics adjuvant carbon ion radiotherapy increases the abundance of Akkermansia muciniphila(AKK),alters the metabolic level of intestinal microbes to regulate the energy and substance metabolism of tumor cells by regulating the metabolism of amino acids,such as tryptophan,arginine,glycine and serine.Additionally,they upregulate the expression of Cd19,Cd79,Fcer2 a,Marco,CD209 b genes in tumor cells to improve the immune level of tumor microenvironment to inhibit the proliferation,metastasis and invasion of tumor.Therefore,the study would provide a promising molecular target and new treatment option for the precise prevention and treatment of breast cancer.(4)The study of the preventive effect of probiotics against CT26 cells induced colon cancer models in mice.When the mice were administrated with probiotics(JY300-8and JMR-01)for 20 days before the injection of CT26 cell in mice,the tumor formation rate and tumor volume were reduced by 86.21% and 65.2%,respectively.The results indicate that probiotics inhibited tumor initiation and progression in colon cancer susceptible mice.Based on 16 S diversity of gut microbiota and metabolomics analysis,the study shows probiotics suppress tumor proliferation and capillarization by downregulating pyrimidine metabolism and sphingosine-1-phosphate(S1P)signaling pathway in tumors.Therefore,our findings suggest that the breeding probiotics have significant value and potential in preventing colon cancer,providing a promising strategy for tumor prevention based on gut microbial therapy. |
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