Gut Dysbiosis Impacts Brain Metastasis Outgrowth and Immune Response Dynamics

We used 16S V3/V4 region amplification to evaluate the composition of bacteria species in mouse fecal pellets after vehicle or ABX treatment and before and after fecal matter transplant. Overall design: To evaluate gut microbiome changes after ABX treatment, fecel pellets were collected from young-adult (12 weeks old) wild type C57Bl/6 mice after 21 days of vehicle or antibiotics treatment (to induce gut microbiota depletion). In one sequencing round, we sequenced a total of 6 different fecal samples (3 control, 3 depleted gut microbiota (ABX)). To evaluate fecal matter transplant (FMT) efficacy, ~12 weeks old C57BL/6 mice were first pre-treated for 14 days with vehicle or ABX. Following the 14-day pre-treatment, all mice were switched to regular water and received PBS via oral gavage for a 2-day washout period. Next, mice from vehicle and ABX pre-treatment groups were randomized and cohoused. For FMT, fresh fecal pellets were collected from mice in the vehicle pre-treatment group. 2-3 pellets were placed in 1 mL of sterile PBS in a 1.5 mL tube and vortexed into a homogenate. The homogenates were then centrifuged at 500 g for 5 minutes and supernatant was collected. All mice received 200 uL of fecal matter supernatant by oral gavage. This procedure was repeated every other day for a seven-day period. Mouse stool was collected post-vehicle/ABX treatment and post FMT/cohouse for 16s rRNA analysis. Mouse fecal DNA extraction was performed using the ZymoBIOMICS DNA Miniprep Kit (Cat# D4300). 16S V3/V4 region amplification, sample indexing and library preparation were performed following the Illumina 16S Metagenomic Sequencing Library Preparation (PN 15044223 Rev. B). Amplicons were indexed using the Nextera XT Index Kit and pooled into a library for Illumina sequencing.