肠道菌群失调对小鼠结肠癌放射免疫治疗远隔效应的干扰

Perturbation of gut microbiota modulated the abscopal effects of immunoradiotherapy for colon cancer in mice

目的研究抗生素处理小鼠肠道菌群对MC38结肠癌放疗联合抗PD-1免疫治疗远隔效应的影响.方法在109只SPF级雌性C57BL/6J小鼠中建立双侧MC38皮下移植肿瘤[原位(放疗部位)肿瘤和远隔肿瘤],给予或不给予口服抗生素治疗.将C57BL/6J小鼠采用完全随机的方式分为8组:对照组(n=15)、放射治疗组(n=17)、抗PD-1免疫治疗组(n=15)、放射联合抗PD-1免疫治疗组(n=17)、单独抗生素组(n=11)、抗生素联合放射治疗组(n=11)、抗生素联合抗PD-1免疫治疗组(n=10)、抗生素联合放射加抗PD-1免疫治疗组(n=13);8组可归为2类:前4组未使用抗生素即正常肠道菌群类(Normal类),后4组使用抗生素即去除肠道菌群类(ATB类).16S rRNA测序小鼠粪便用于评估ATB后肠道菌群的改变.利用多种生物信息学方法研究特定肠道菌群对远隔效应的影响.结果原位肿瘤在经过放射联合抗PD-1免疫治疗后,Normal类和ATB类小鼠均表现出肿瘤缩小,均P<0.001.在Normal类中,放射联合抗PD-1免疫治疗组远隔肿瘤的大小较其他3组均减小,差异均有统计学意义,均P<0.05.ATB类中,抗生素联合放射加抗PD-1免疫治疗组远隔肿瘤的大小较其他3组差异无统计学意义,均P>0.05.ATB后肠道菌群中α-多样性显著降低,Nor-mal类和ATB类菌群的β-多样性存在差异.线性判别分析(LEfSe)结果显示,Normal类显著差异物种为p_Firmicutes和g_Ligilactobacillus,ATB类显著差异物种为p_Bacteroidota和g_Bacteroides.在门水平,使用ATB后,拟杆菌门的相对丰度显著升高(P<0.001),厚壁菌门的相对丰度显著降低(P<0.001);在属水平,ATB后,拟杆菌属的相对丰度显著升高(P<0.001),唾液乳杆菌的相对丰度显著降低,P<0.001.结论抗生素治疗对肠道菌群的改变,可显著影响小鼠MC38结肠癌的放射联合抗PD-1免疫治疗的远隔效应.

Objective To investigate whether the gut microbiota of mice treated with antibiotics has an effect on the distant effect of MC38 colon cancer radiotherapy combined with anti-PD-1 immunotherapy in mice.Methods Bilateral MC38 subcutaneous grafted tumors[in situ(radiotherapy site)tumors and distant tumors]were established with or without oral antibiotic therapy in 109 SPF grade female C57BL/6J mice.C57BL/6J mice were divided into 8 groups in a completely random way:control group(n=15),radiotherapy group(n=17),anti-PD-1 immunotherapy group(n=15),radiothera-py combined with anti-PD-1 immunotherapy group(n=17),antibiotics alone group(n=11),antibiotics combined with radiotherapy group(n=11),antibiotics combined with anti-PD-1 immunotherapy group(n=10),and antibiotics combined with radiotherapy anti-PD-1 immunotherapy group(n=13).The eight groups could be classified into two categories,the first 4 groups did not use antibiotics,which were normal intestinal flora(Normal),and the last 4 groups used antibiotics which were eliminated intestinal flora(ATB).16S rRNA sequencing of mouse feces was used to assess changes in the gut microbiome after ATB.A variety of bioinformatics methods were used to investigate the effects of spe-cific gut flora on specific flora associated with distant effects.Results In situ tumor after radiotherapy combined with an-ti-PD-1 immunotherapy,both Normal and ATB mice showed tumor shrinkage(P<0.001).In the Normal group,the size of distant tumors in the radiotherapy combined with anti-PD-1 immunotherapy group decreased compared with the other 3 groups,with statistical significance,all P<0.05.In the ATB group,there was no significant difference in the size of distant tumors in the antibiotic combined with radiation plus anti-PD-1 immunotherapy group compared with the other 3 groups,all P>0.05.The a-diversity of gut microbiota decreased significantly after ATB,and theβ-diversity be-tween Normal and ATB microbiota was significantly different.The results of linear discriminant analysis effect size(LEfSe)showed that the species with significant difference in Normal group was p Firmicutes and g_Ligilactobacillus,while the species with significant difference in ATB group was p_Bacteroidota and g_Bacteroides.At the phylum level,the relative abundance of Bacteroidota increased significantly(P<0.001)and the relative abundance of Firmicutes is sig-nificantly reduced(P<0.001)after ATB.At the genus level,after ATB,the relative abundance of Bacteroides increased significantly(P<0.001),and the relative abundance of Lactobacillus salivary significantly decreased(P<0.001)after ATB.Conclusion The changes of gut microbiota after ATB significantly affect the abscopal effects of iRT for colon cancer.