Lactobacillus Acidophilus Enhanced PD-1 Inhibitors Antitumor-immunity In A Lewis Lung Cancer Mouse Model After Antibiotics Treatment

[Background]Patients with advanced malignant tumors are more susceptible to have bacterial infections due to their own characteristics and iatrogenic injuries.However,long-term antibiotic can sharply reduce the abundance of intestinal microbiota and alter the composition of gut commensals,futher impacting host’s immune responses and subsequently diminishing the efficacy of immune-checkpoint inhibitors(ICIs)therapy.Probiotics,as live bacteria that are beneficial to the host,one of the beneficial mechanisms is to restore and regulate the gut microbiota.Lactobacillus acidophilus(LAC)is one of the most representative species as probiotics,it can develop a protective barrier on the surface of intestinal mucosa and reduce the intestinal pH to create an environment that is not conducive to the growth of harmful bacteria,which in turn regulate the gut microbiota.Therefore,timely supplement of LAC after antibiotic can help normalize the gut microbiota and promote the efficacy of anti-tumor immunotherapy.[Objective]1.To explore the relationship between the gut microbiota and the clinical prognosis of ICIs treatment in NSCLC patients through meta-analysis.2.By constructing a Lewis lung cancer(LLC)mouse model,the impact of antibiotic-mediated gut microbiota disturbance on the efficacy of PD-1 monoclonal antibody was confirmed.3.By constructing a antibiotic-treated LLC mouse model,the repair effect of LAC on intestinal microbiota imbalance was confirmed,and the impact and potential mechanism of LAC on PD-1 monoclonal antibody therapy were explored.[Methods]1.By retrieving published articles,we extracted the taxa data and classified them according to the relationship of ICIs treatment response,then constructed a phylogenetic tree.Besides,we analyzed the pooled effect size of a diversity.2.LLC mice were randomly divided into non antibiotic(non-ATB)group and antibiotic(ATB)groups before anti-PD-1 treatment,a mouse in ATB groups was fed with antibiotic cocktail of ampicillin,metronidazole,neomycin and vancomycin every day for a week,non-ATB received mice were given the equal amount of normal saline.During anti-PD-1 therapy,we recorded tumor sizes and collected mice feces for test.3.LLC mouse model post-antibiotics received four types of treatment regiments,namely:homotypic control group,anti-PD-1 group,LAC group and anti-PD-1+LAC group.LAC were administered to mice by daily oral gavage everyday after tumor detection.We recorded the change of tumor sizes,analyzed the characteristics of the gut microbiota in each group at the end of the experiment,and explore the mechanism by using flow cytometry analysis,immunohistochemistry analysis,and quantitative real-time polymerase chain reaction.[Results]1.Bifidobacterium,Faecalibacterium,Ruminococcus,Alistipes and Prevotella were positively correlated with ICIs treatment response in NSCLC patients.The a-diversity defined by richness and evenness was strongly associated with the efficacy of ICIs treatment.2.Antibiotics use reduced the inhibitory effect of PD-1 monoclonal antibody on tumor growth,and significantly lowered the a diversity of the gut microbiota and the relative abundance of beneficial bacteria for ICIs treatment.3.LAC combined with anti-PD-1 mAb enhanced anti-tumor immunity in LLC mouse model post-antibiotics,it restored the α diversity and significantly increased the relative abundance of beneficial bacteria(Bifidobacterium,Faecalibacterium,Ruminococcus,Alistipes and Prevotella)for ICIs therapy.The mechanism were reducing the proportion of myeloid-derived suppressor cells and the secretion of IL-10 and TGF-β in the spleen,activating CD8+T cells and the of memory T cells and increasing the level of TNF-α,inhibiting the polarization of M2-type tumor-associated macrophages and reduces the production of IL-10 and TGF-β in the tumor microenvironment.[Conclusion]Antibiotics administration might attenuate the inhibitory effect of PD-1 inhibitors on tumor growth by inducing intestinal flora disturbance.LAC combined with anti-PD-1 monoclonal antibody enhanced anti-tumor immunity by regulating intestinal microbiota and synergistically improving anti-tumor immunity,which in turn significantly suppressed the tumor growth.We provided more evidence and possibilities for the anti-tumor immunotherapy function of the gut microbiota.