Intrastromal Therapy In-sync with Featured Stereotactic Body Radiotherapy (SBRT)-Fixing the Defiant Tumor Microenvironment-A Proof of Concept Study

Abstract

We sought to determine whether specific taxa in the communities of the C57BL/6J mouse intestinal microbiome were associated with survival after the LD 50/30 dose of total body irradiation (TBI), and if administration of a second-generation probiotic producing anti-inflammatory Interleukin-22 (IL-22) mitigated Gastrointestinal Syndrome inducing doses of total body irradiation (TBI). Materials/Methods: Female C57BL/6J mice were irradiated to LD 50/30 9.25 Gy TBI, or 19.75 Gy whole abdominal irradiation, and evaluated for primary endpoint of survival and secondary endpoint of expression of inflammatory proteins and bone marrow CFU-GEMMs per 10 4 cells. Daily collected fecal samples were analyzed for 16sRNA associated with 15 major taxa in the intestinal microbiome. Second-generation probiotic Lactobacillus reuteri or Escherichia-coli producing IL-22 (LR-IL-22 or E. coli-IL-22, respectively) was administered to subgroups by gavage at 24 hours after irradiation. The results were compared to subcutaneous administration of IL-22 (n Z 12). Results: Thirty-day TBI survivors of 9.25 Gy had at day 14 a predictive increased relative abundance of Lactobacillus, Roseburia, and Akkermansia, compared to other taxa, as did radiation mitigator treated (G-CSF or JP4-039) mice. Administration of LR-IL-22 at 24 hours after irradiation increased survival (p Z 0.0144), as did E. coli-IL-22. GFP-IL-22 fusion protein producing probiotics showed uptake in intestinal villi at 2 h after gavage and clearance by day 5. At 24 hrs. after 9.25 Gy TBI, mice with 4 antibiotic-cleared intestinal microbiomes (5 weeks administration in drinking water of vancomycin, neomycin trisulfate, metronidazole, and ampicillin) had increased survival when treated with E. coli-IL-22, (p < 0.0001). On day 5 after 9.25 Gy TBI, the intestine and bone marrow were isolated. Luminex assay showed significantly decreased inflammatory proteins in mice gavaged with LR-IL-22, and bone marrow had significantly increased CFU-GEMMs per 10 4 cells compared to 9.25 Gy only (15.1 AE 1.1 and 9.1 AE 1.5, respectively, p Z 0.0351). Whole abdomen irradiation to 19.75 Gy followed by gavage at 24 hrs. of LR-IL-22, or E. coli-IL-22 significantly increased survival (p Z 0.0138, 0.0473). Conclusion: These data indicate that the relative abundance of specific taxa in the intestinal microbiome correlates with survival after total body irradiation. Furthermore, gavage of LR-IL-22 improves survival after GI Syndrome inducing doses of irradiation. Elucidation of the molecular mechanism of interaction of pro-survival microbiome communities with intestinal stem cells and regenerating crypts should identify new targets for intestinal radiation protection and mitigation.