Perturbation of Gut Microbiota Modulated the Abscopal Effects of Immunoradiotherapy in Rectal Cancer

Abstract

The abscopal effect-the regression of malignancies outside the irradiation zone-can be increased by combining radiotherapy (RT) with immunotherapy. In this study, we aimed to investigate whether the gut microbiota affected the abscopal effect following immunoradiotherapy (iRT) in rectal cancer. Bilateral MC38 subcutaneous tumors (primary and abscopal tumor) were established in C57/B6 mice with or without oral antibiotic treatment (preadministration of vancomycin, streptomycin, and ampicillin 14 days before therapy). Mice with or without antibiotic therapy were then randomized into eight groups to receive one of four treatments: (1) RT to the primary tumor + anti-PD-1 therapy (iRT), (2) RT to the primary tumor (RT), (3) anti-PD-1 therapy (anti-PD-1), and (4) no treatment. Flow cytometry was used to determine the composition and function of immune cells in the primary and abscopal tumors as well as in the spleen. 16S rRNA sequencing was used to assess the gut microbiome alteration following antibiotic intervention. Multiple bioinformatics were then explored to investigate the impact of specific flora that related to abscopal antitumor effect. We found that radiation on primary tumors exhibited cytotoxic effect in nonirradiated (abscopal) tumors (p=0.0057, RT vs. untreated group; p=0.0037, iRT vs. anti-PD-1 group). In contrast, abscopal tumors were resistant to the anticancer effects of RT when antibiotics were given (p=0.5374, RT + antibiotics vs. untreated group + antibiotics; p=0.42, iRT + antibiotics vs. anti-PD-1 + antibiotics group). Comparing the RT+antibiotics group to the RT group, we discovered that the number of CD8+CD44+ T cells decreased significantly in both abscopal tumors (p<0.001) and spleens (p=0.0061). In anti-PD-1-treated groups, antibiotics significantly reduced the number of CD8+GranzymeB+ T cells in primary tumors (p=0.0061) and CD4+CD25+ T cells in spleens (p<0.001). In iRT-treated groups, the antibiotic reduced the fraction of CD4+INF-γ+ cells in abscopal tumors (p=0.0134), and increased the number of CD4+PD-1+ T cells in spleens (p<0.001). Moreover, we found that both α- and β-diversity decreased significantly in the gut microbiota after antibiotic treatment (p=0.0079 and p<0.001, respectively). The abundance of g_Alistipes, g_Lactobacillus, g_Lachnospiraceae and g__Lactobacillus fell dramatically in the presence of antibiotics. In addition, functional analyses of Picrust2 and KEGG revealed that antibiotic therapy had the most profound impact on the D-Alanine metabolism pathway (p<0.001). We found that the alteration of the gut microbiome by antibiotics significantly affects the local and systemic antitumoral effect of iRT, our results may provide new insight on how gut modification converts the local anticancer effects of RT into a systemic response that targets metastatic tumors.